Method for targeted delivery of nucleic acids

ABSTRACT

The present invention is directed to a method of in vivo and ex vivo gene delivery, for a variety of cells. More specifically, it relates to a novel carrier system and method for targeted delivery of nucleic acids to mammalian cells. More specifically, the present invention relates to carrier system comprising single-chain polypeptide binding molecules having an a region rich in basic amino acid and having the three dimensional folding and, thus, the binding ability and specificity, of the variable region of an antibody. The basic amino acid rich region can comprise oligo-lysine, oligo-arginine or combinations thereof. Such preparations of modified single chain polypeptide binding molecules also have ability to bind nucleic acids at the region rich in basic amino acid residues. These properties of the modified single chain polypeptide binding molecules make them very useful in a variety of therapeutic applications including gene therapy. The invention also relates to multivalent antigen-binding molecules having regions rich in basic amino acids. Compositions of, genetic constructions for, methods of use, and methods for producing basic amino acid tailed antigen-binding proteins are disclosed.

The present application claims benefit of the filing date of U.S.application Ser. No. 60/104,949, filed Oct. 20, 1998, which disclosureis incorporated herein in entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method of in vivo and ex vivogene delivery, for a variety of cells. More specifically, it relates toa novel carrier system and method for targeted delivery of nucleic acidsto mamimalian cells. More specifically, the present invention relates tocarrier systems comprising single-chain polypeptide binding moleculeshaving a basic amino acid rich region, such as an oligo-lysine or anoligo-arginine region, and having the three dimensional folding and,thus, the binding ability and specificity, of the variable region of anantibody. Such preparations of modified single chain polypeptide bindingmolecules also have ability to bind nucleic acids at the basic aminoacid rich region. These properties of the modified single chainpolypeptide binding molecules make them very useful in a variety oftherapeutic applications including gene therapy. The invention alsorelates to multivalent antigen-binding molecules having basic amino acidrich regions. Compositions of, genetic constructions for, methods ofuse, and methods for producing such basic amino acid rich regioncontaining antigen-binding proteins are disclosed.

2. Description of Related Art

Substantial attention has been given to the promise of gene therapy inrecent years. This term has been used to describe a wide variety ofmethods using recombinant biotechnology techniques to deliver a varietyof different materials to a cell. Such methods include, for example, thedelivery of a gene, antisense RNA, a cytotoxic agent, etc., by a vectorto a mammalian cell, preferably a human cell either in vivo or ex vivo.Most of the initial work has focused on the use of retroviral vectors totransform these cells. This focus has resulted from the ability ofretroviruses to infect cells with high efficiency.

However, numerous difficulties with retroviruses have been reported. Forexample, problems have been encountered in infecting certain cell types.Retroviruses typically enter cells via receptors and if such receptorsare not present on the cell, or not present in large numbers, theninfection is not possible or efficient. These viruses are alsorelatively labile in comparison to other viruses. Outbreaks of wild-typevirus from recombinant virus-producing cell lines have also beenreported with the vectors themselves causing disease. Moreover, theseviruses are only expressed in dividing cells.

In addition, retroviral-mediated gene transfer methods typically resultin stable transformation of the target cells. Although this may beregarded as advantageous, the stable transformation of a patient'ssomatic cells makes it difficult to reverse the treatment regimen ifundesirable side effects occur. Moreover, there is the concern thatgenetic transformation might lead to malignant transformation of thecell.

Other methods of delivering genetic material to cells in vivo and exvivo include the use of liposome entrapped DNA. Liposomes are smallmembrane-enclosed spheres that have been formed with the appropriate DNAentrapped within it. However, this system also has inherent problems. Itis difficult to control the size of the liposome and, hence theuniformity of delivery to individual cells. Additionally, it isdifficult to prevent leakage of the contents of the liposomes and aswith other techniques, there has been difficulty in directing cell-typespecificity.

Antibodies are proteins generated by the immune system to provide aspecific molecule capable of complexing with an invading molecule,termed an antigen. Natural antibodies have two identical antigen-bindingsites, both of which are specific to a particular antigen. The antibodymolecule “recognizes” the antigen by complexing its antigen-bindingsites with areas of the antigen termed epitopes. The epitopes fit intothe conformational architecture of the antigen-binding sites of theantibody, enabling the antibody to bind to the antigen. The antibodymolecule is composed of two identical heavy and two identical lightpolypeptide chains, held together by interchain disulfide bonds. Theremainder of this discussion on antibodies will refer only to one pairof light/heavy chains, as each light/heavy pair is identical. Eachindividual light and heavy chain folds into regions of approximately 110amino acids, assuming a conserved three-dimensional conformation. Thelight chain comprises one variable region (V_(L)) and one constantregion (C_(L)), while the heavy chain comprises one variable region(V_(H)) and three constant regions (C_(H)1, C_(H)2 and C_(H)3). Pairs ofregions associate to form discrete structures. In particular, the lightand heavy chain variable regions associate to form an “Fv” area whichcontains the antigen-binding site. The constant regions are notnecessary for antigen binding and in some cases can be separated fromthe antibody molecule by proteolysis, yielding biologically active(i.e., binding) variable regions composed of half of a light chain andone quarter of a heavy chain.

Further, all antibodies of a certain class and their F_(ab) fragments(i.e., fragments composed of V_(L), C_(L), V_(H), and C_(H)1) whosestructures have been determined by x-ray crystallography show similarvariable region structures despite large differences in the sequence ofhypervariable segments even when from different animal species. Theimmunoglobulin variable region seems to be tolerant towards mutations inthe antigen-binding loops. Therefore, other than in the hypervariableregions, most of the so-called “variable” regions of antibodies, whichare defined by both heavy and light chains, are, in fact, quite constantin their three dimensional arrangement. See for example, Huber, R.,Science 233:702-703 (1986).

Recent advances in immunobiology, recombinant DNA technology, andcomputer science have allowed the creation of single polypeptide chainmolecules that bind antigen. These single-chain antigen-bindingmolecules (“SCA”) or single-chain variable fragments of antibodies(“sFv”) incorporate a linker polypeptide to bridge the individualvariable regions, V_(L) and V_(H), into a single polypeptide chain. Adescription of the theory and production of single-chain antigen-bindingproteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203,5,455,030 and 5,518,889. The single-chain antigen-binding proteinsproduced under the process recited in the above U.S. patents havebinding specificity and affinity substantially similar to that of thecorresponding Fab fragment. A computer-assisted method for linker designis described more particularly in Ladner et al., U.S. Pat. Nos.4,704,692 and 4,881,175, and WO 94/12520.

The in vivo properties of sFv polypeptides are different from MAbs andantibody fragments. Due to their small size, sFv polypeptides clear morerapidly from the blood and penetrate more rapidly into tissues (Milenic,D. E. et al., Cancer Research 51:6363-6371 (1991); Colcher et al., J.Natl. Cancer Inst. 82:1191 (1990); Yokota et al., Cancer Research52:3402 (1992)). Due to lack of constant regions, sFv polypeptides arenot retained in tissues such as the liver and kidneys. Due to the rapidclearance and lack of constant regions, sFv polypeptides will have lowimmunogenicity. Thus, sFv polypeptides have applications in cancerdiagnosis and therapy, where rapid tissue penetration and clearance, andease of microbial production are advantageous.

A multivalent antigen-binding protein has more than one antigen-bindingsite. A multivalent antigen-binding protein comprises two or moresingle-chain protein molecules. Enhanced binding activity, di- andmulti-specific binding, and other novel uses of multivalentantigen-binding proteins have been demonstrated. See, Whitlow, M., etal., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R., NatureBiotech. 15:125-126 (1997); and WO 93/11161.

Ladner et al. also discloses the use of the single chain antigen bindingmolecules in diagnostics, therapeutics, in vivo and in vitro imaging,purifications, and biosensors. The use of the single chain antigenbinding molecules in immobilized form, or in detectably labeled forms isalso disclosed, as well as conjugates of the single chain antigenbinding molecules with therapeutic agents, such as drugs or specifictoxins, for delivery to a specific site in an animal, such as a humanpatient.

Whitlow et al. (Methods: A Companion to Methods in Enzymology2(2):97-105 (June, 1991)) provide a good review of the art of singlechain antigen binding molecules and describe a process for making them.

In U.S. Pat. No. 5,091,513, Huston et al. discloses a family ofsynthetic proteins having affinity for preselected antigens. Thecontents of U.S. Pat. No. 5,091,513 are incorporated by referenceherein. The proteins are characterized by one or more sequences of aminoacids constituting a region that behaves as a biosynthetic antibodybinding site (BABS). The sites comprise (1) noncovalently associated ordisulfide bonded synthetic V_(H) and V_(L) regions, (2) V_(H)-V_(L) orV_(L)-V_(H) single chains wherein the V_(H) and V_(L) are attached to apolypeptide linker, or (3) individual V_(H) or V_(L) domains. Thebinding domains comprises complementarity determining regions (CDRs)linked to framework regions (FRs), which can be derived from separateimmunoglobulins.

U.S. Pat. No. 5,091,513 also discloses that three subregions (the CDRs)of the variable domain of each of the heavy and light chains of nativeimmunoglobulin molecules collectively are responsible for antigenrecognition and binding. These CDRs consist of one of the hypervariableregions or loops and of selected amino acids or amino acid sequencesdisposed in the framework regions that flank that particularhypervariable region. It is said that framework regions from diversespecies are effective in maintaining CDRs from diverse other species inproper conformation so as to achieve true immunochemical bindingproperties in a biosynthetic protein.

U. S. Pat. No. 5,091,513 includes a description of a chimericpolypeptide that is a single chain composite polypeptide comprising acomplete antibody binding site. This single chain composite polypeptideis described as having a structure patterned after tandem V_(H) andV_(L) domains, with a carboxyl terminal of one attached through an aminoacid sequence to the amino terminal of the other. It thus comprises anamino acid sequence that is homologous to a portion of the variableregion of an immunoglobulin heavy chain (V_(H)) peptide bonded to asecond amino acid sequence that was homologous to a portion of thevariable region of an immunoglobulin light chain (V_(L).)

Chen et al., describe the production and use of a fusion proteinconsisting of an antibody Fab fragment and a DNA binding moiety,protamine, to deliver toxin-expressing plasmid DNA into HIV infectedcells by receptor mediated endocytosis (S-Y Chen et al., Gene Therapy 2:116-123 (1995)).

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a newand improved delivery system that can introduce foreign genes in anon-toxic, cell specific manner into mammalian cells. Also provided bythe invention is a system and an efficient method that exhibits a highdegree of cell specificity using relatively simple yet reliabledelivery.

Another feature of the present invention is the use of receptor-mediatedspecificity to provide cell specificity to the gene delivery system.This involves the use of cell-surface receptors as naturally existingentry mechanisms for the specific delivery of genes. The molecules oncerecognized and bound to the receptor can be internalized within thetarget cell via endocytosis. Included in this feature is the provisionfor a unique carrier comprising a single-chain antigen-bindingprotein/polynucleotide complex capable of targeting the gene to specificcells possessing particular receptors that recognize the complex.

In addition, the carrier of the present invention relates to tailedsingle chain polypeptides containing a basic amino acid rich region(i.e., oligo-lysine, oligo-arginine, or a mixture thereof) and havingbinding affinity for an antigen and the capability of delivering nucleicacids to a cell and processes for preparing them. Suitable polypeptidesare, for example, those described by Ladner et al. in U.S. Pat. No.4,946,778 and Huston et al. in U.S. Pat. No. 5,091,513.

These features provide advantages to the present invention that directlycontribute to the efficiency and target specificity of the deliverysystem to specific cell types, including normal cells as well as tumorcells not found in the delivery systems known in the art.

The present invention is directed to a method of delivering nucleicacids to a cell comprising:

(1) providing an a basic amino acid tailed single-chain antigen-bindingpolypeptide capable of delivering nucleic acids to a cell comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein, at its C-terminus, N-terminus, or both of polypeptide (a), (b)or both, the single-chain antigen-binding polypeptide has an amount ofbasic amino acid residues sufficient to bind nucleic acids, wherein thebasic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen;

(2) allowing a nucleic acid to bind to the basic amino acid residuecontaining single-chain antigen-binding polypeptide; and

(3) transforming a cell with the nucleic acid bound basic amino acidresidue containing single-chain antigen-binding polypeptide.

More particularly, the invention is directed to a single-chainantigen-binding polypeptide capable of delivering nucleic acids to acell, comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b)or both, the single-chain antigen-binding polypeptide has an amount ofbasic amino acid residues sufficient to bind nucleic acids, wherein thebasic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen. These basic amino acid residues in the sFvprotein (e.g., oligo-lysine sFv) generate a minimal non-specific nucleicacid binding region. The basic amino acid region is configured such thatat least 2 to 8 groups of eight consecutive residues of Lys, Arg or acombination thereof are separated from adjacent groups by 0-20 aminoacid residues.

The invention is further directed to a genetic sequence encoding asingle-chain antigen-binding polypeptide capable of delivering nucleicacids to a cell, comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein at its C-terminus, N-terminus, or both of polypeptide (a),(b) orboth, the single-chain antigen-binding polypeptide has an amount ofbasic amino acid residues sufficient to bind nucleic acids, wherein thebasic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen. These basic amino acid residues in the sFvprotein (e.g., oligo-lysine sFv) generate a minimal non-specific nucleicacid binding region. The basic amino acid region is configured such thatat least 2 to 8 groups of eight consecutive residues of Lys, Arg or acombination thereof are separated from adjacent groups by 0-20 aminoacid residues.

The nucleic acid is a polynucleotide that can be either DNA or RNA.

The invention is directed to a replicable cloning or expression vehiclecomprising the above described polynucleotide sequence. The invention isalso directed to such vehicle which is a plasmid. The invention isfurther directed to a host cell transformed with the above describedDNA. The host cell can be a bacterial cell, a yeast cell or other fungalcell, an insect cell or a mammalian cell line. A preferred host isPichia pastoris.

The invention is directed to a method of producing a single-chainantigen-binding polypeptide capable of delivering nucleic acids to acell, comprising:

(a) providing a first genetic sequence encoding a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain;

(b) providing a second genetic sequence encoding a second polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; and

(c) linking the first and second genetic sequences (a) and (b) with athird genetic sequence encoding a peptide linker into a fourth geneticsequence encoding a single chain polypeptide having an antigen bindingsite, wherein at its C-terminus, N-terminus, or both of polypeptide (a),(b) or both, the single-chain antigen-binding polypeptide has an amountof basic amino acid residues sufficient to bind nucleic acids, whereinthe basic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen;

(d) transforming a host cell with the fourth genetic sequence encoding asingle-chain antigen-binding polypeptide of (c); and

(e) expressing the single-chain antigen-binding polypeptide of (c) inthe host, thereby producing a single-chain antigen-binding polypeptidecapable of delivering nucleic acids to a cell.

The invention is further directed to a multivalent single-chainantigen-binding protein, comprising two or more single-chainantigen-binding polypeptides, each single-chain antigen-bindingpolypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b)or both, the single-chain antigen-binding polypeptide has an amount ofbasic amino acid residues sufficient to bind nucleic acids, wherein thebasic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen.

In the above described embodiments of the invention, a lysine rich or anoligo-Lys polypeptide sequence of the present invention can be capableof attaching apolyalkylene oxide moiety wherein the polyalkylene oxideconjugated oligo-lysine tailed single-chain antigen-binding polypeptidebinds an antigen as well as nucleic acids.

In the above described embodiments of the invention, the C-terminus ofthe second polypeptide (b) can be the native C-terminus. The C-terminusof the second polypeptide (b) can comprise a deletion of one orplurality of amino acid residue(s), such that the remaining N-terminusamino acid residues of the second polypeptide are sufficient for thepolypeptide to be capable of binding an antigen. The C-terminus of thesecond polypeptide can comprise an addition of one or plurality of aminoacid residue(s), such that the polypeptide is capable of binding anantigen. Moreover, the nucleic acid binding region can be generated bymutating one or a plurality of amino acid residue(s) to a basic aminoacid residue(s) in the C-terminal or N-terminal regions of thepolypeptide (a) or (b). In addition, the nucleic acid binding region canbe generated by inserting blocks of basic amino acids at the C-terminusor N-terminus of the polypeptide (a) or (b).

In a preferred embodiment of the invention, the first polypeptide (a)can comprise the antigen binding portion of the variable region of anantibody light chain and the second polypeptide (b) comprises theantigen binding portion of the variable region of an antibody heavychain.

The invention is also directed to a method for treating a targeteddisease, comprising administering an effective amount of a compositioncomprising a nucleic acid molecule bound to the polypeptide or proteinof the invention and a pharmaceutically acceptable carrier vehicle fordelivery to a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA (SEQ ID NO:1) and protein sequence (SEQ ID NO:2) ofCC49/218 sFv with an engineered oligo-lysine C-terminal tail segment.The eight new lysine residues were genetically engineered at a BstEIIsite and are shown underlined and marked with asterisks. Alsohighlighted are the CDR sequences (double underlined), the 218 linker(underlined and labeled) and selected restriction sites.

FIGS. 2A and 2B show the DNA (SEQ ID NO:3) and protein sequence (SEQ IDNO:4) of CC49/218 sFv with an engineered oligo-lysine C-terminal tailsegment. The sixteen new lysine residues were genetically engineered ata BstEII site and are shown underlined and marked with asterisks. Alsohighlighted are the CDR sequences (double underlined), the 218 linker(underlined and labeled) and selected restriction sites.

FIG. 3 shows the protein sequence (SEQ ID NO:5) of A33/218 sFv withengineered oligo-lysine C-terminal tail segment. The sixteen new lysineresidues are marked with asterisks. Also highlighted are the CDRsequences (double underline) and the 218 linker (overlined and labeled).

FIG. 4 shows DNA binding by A33/218 SCA with an engineered 16 lysineC-terminal tail using gel shift assay: lane 1 is a BSA control, lane 2is a GS115 culture supernatant control and lanes 3-12 have 0, 5, 10, 15,20, 30, 40, 50, 60, 70 and 80 μl, respectively of dialyzed culturesupernatant of the 16 lysine SCA protein.

FIG. 5 shows the Coomassie Blue stained SDS-PAGE gel of purifiedCC49-16K 266(7). Lane 1, molecular weight markers; Lane 2, purifiednative CC49/218 sFv; Lane 3, EN266(7) fermentation cell pellet; Lane 4,EN266(7) sFv released from Lane 3 material by a high salt wash (1.5 MNaCl, 20 mM Tris-HCl, pH 8.0, at room temperature for 2 hours).

FIG. 6 shows an ELISA assay demonstrating retention of mucin-bindingactivity of the CC49-16K sFv EN266(7).

FIGS. 7A and 7B show the results of the transfection of LS 174-T cellsby reporter plasmid pSEAP using CC49-16K sFv as carrier.

FIG. 8 shows the sequence for Kabat Consensus V_(k)I/218/V_(H)III sFv(SEQ ID NO:6). The sixteen new lysine residues are marked withasterisks. CDR sequences are double underlined.

FIG. 9 shows the sequence for C6.5/218 sFv (SEQ ID NO:7). The sixteennew lysine residues are marked with asterisks. CDR sequences are doubleunderlined.

DESCRIPTION OF THE EMBODIMENTS

The present invention is directed to the novel combination of asingle-chain polypeptide containing basic amino acid region (e.g.,regions rich in basic amino acids, oligo-lysine, oligo-arginine orcombination thereof) and having 1) capability to bind nucleic acids; and2) binding affinity for an antigen, such that the polypeptide is capableof delivering nucleic acids to cells. The present invention is alsodirected to a method of delivering nucleic acids to cells using a basicamino acid tailed single chain polypeptide. Furthermore, the foregoingdesign could applied not only to sFvs but also to V_(H) single domains,disulfied-stabilized Fv, Fabs or Mabs.

Gene Delivery Methods

The present invention provides a novel delivery system that canintroduce foreign genes in a non-toxic, cell specific manner intomammalian cells ex vivo or in vivo. Also provided by the invention is asystem and method that exhibits a high degree of cell specificity usingrelatively simple yet reliable delivery.

The present invention uses a receptor-mediated specificity to providecell specificity to the gene delivery system. This involves the use ofcell-surface receptors or antigens as naturally existing entrymechanisms for the specific delivery of genes. Included in this featureis the provision for a unique a basic amino acid tailed single-chainantigen-binding polypeptide polynucleotide complex capable of targetingthe gene to specific cells possessing particular receptors or antigensthat are recognized by the complex. Cell specificity can be achieved byselecting a single-chain antigen-binding protein that has a bindingaffinity for the cell type to be targeted for gene delivery. Forexample, anti-tumor single-chain antigen-binding protein can be used totarget gene delivery to specific tumor cells. Also, anti-fluoresceinsingle-chain antigen-binding proteins can be used to target fluoresceinlabeled cells. Thus, the skilled artisan could readily target any celltype by selecting a single-chain antigen-binding protein having anappropriate affinity for the targeted cell.

In addition, the cell specificity for the targeted delivery of nucleicacids can be achieved or enhanced by including “translocation domains”in the sFvs of the present invention. The use of the exotoxin A“translocation domain” has been demonstrated to facilitate efficient DNAtransfer in non-viral DNA delivery systems. See, Fominaya et al. J.Biol. Chem. 271: 10560 (1986); and WO 96/13599, incorporated byreference). Also, nucleus targeting peptide fusions have demonstratedenhanced delivery of DNA to the nucleus in non-viral DNA deliverysystems. (See, Avrameas et al. Proc. Natl. Acad. Sci. 95: 5601-5606(1998)). Thus, the skilled artisan could readily further enhance theefficiency nucleic acid delivery to a target cell type by including“translocation domain” and or a nucleus targeting peptide within asingle-chain antigen-binding protein which has an appropriate affinityfor the targeted cell.

The present invention is directed to a method of delivering nucleicacids to a cell comprising:

(1) providing an basic amino acid tailed single-chain antigen-bindingpolypeptide capable of delivering nucleic acids to a cell comprising:

(a) a first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and(b) into a single chain polypeptide having an antigen binding site,wherein at its C-terminus, N-terminus, or both of polypeptide (a), (b)or both, the single-chain antigen-binding polypeptide has an amount ofbasic amino acid residues sufficient to bind nucleic acids, wherein thebasic amino acid residues are selected from the group consisting of:Lys, Arg and a combination thereof; and wherein the basic amino acidresidues binds nucleic acid and wherein the single-chain antigen-bindingpolypeptide binds antigen;

(2) allowing a nucleic acid to bind to the basic amino acid tailedsingle-chain antigen-binding polypeptide; and

(3) transforming a cell with the nucleic acid bound basic amino acidtailed single-chain antigen-binding polypeptide.

The invention also provides for the use of the basic amino acid tailedsFv proteins (i.e., oligo-Lys, oligo-Arg or oligo combination of lys andarg residues; or regions rich in lys and/or arg residues) in a processfor targeted gene therapy. The invention further provides for sFvproteins having an amount of basic amino acid residues sufficient tobind nucleic acids. More specifically, the invention provides for sFvproteins having at least 10, 12, 14 or 16 lysines in the C-terminalregion of the sFv which bind nucleic acids wherein the lysine residuesare configured in two groups of eight consecutive lysine residuesseparated by 0-20 amino acid residues. A 16 lysine C-terminal tailed SCAprotein complexed to a nucleic acid construct capable of expressing aprotein, can be used to deliver such nucleic acid constructs to aspecific cell type for (1) transient expression of the protein or (2)allow for the DNA construct to be inserted safely into the genome andhave the expression be regulated by normal cellular signals. To targetthe nucleic acid delivery to a specific cell type, an SCA protein isselected that will bind to and be internalized by that cell type. Manysuch SCA proteins are well known to those skilled in the art. Forexample, anti-tumor SCAs can be modified to have a 16 lysine C-terminaltail according to the present invention. These anti-tumor SCAs can beused to carry DNA encoding toxins or chemotherapeutic proteins that,when internalized and expressed, will cause the death of the tumor cell.

Furthermore, PEGylating the oligo-lysine containing SCA protein oraccording to the methods disclosed in U.S. application Ser. No.091069,842, filed on Apr. 30, 1998 (incorporated by reference in itsentirety), will also provide protection from degradation for thecomplexed nucleic acid since the modified SCA proteins have reducedimmunogenicity and antigenicity as well a longer half-life in thebloodstream. In addition, an SCA protein having one or more lysineresidues in a basic amino acid rich region will allow for site specificPEGylation at the lysine residue(s).

As indicated above, the single-chain antigen-binding polypeptides have anucleic acid binding region comprising a sufficient amount of basicamino acids to bind nucleic acids. This region can comprise a sequencethat is rich in basic amino acids such as lysine, arginine andcombinations thereof This region will contain enough basic amino acidsto obtain the requisite overall positive charge on the sFv for nucleicacid binding. These nucleic acid binding regions can be at theC-terminal region, N-terminal region or both of the sFv. The nucleicacid binding regions can be generated by mutating one or a plurality ofamino acid residue(s) of the sFv or by adding a block of basic aminoacid residues to the C-terminal region, N-terminal region or both of thesFv. Furthermore, the foregoing design could applied not only to sFvsbut also to V_(H) single domains, disulfide-stabilized Fv, Fabs or Mabs.

Preferably, the single-chain antigen-binding polypeptide according tothe present invention has an amount of oligo-Lys, oligo-Arg oroligo-Lys/Arg residues sufficient to bind nucleic acids. Preferably, thenucleic acid binding region of single-chain antigen-binding polypeptidecomprises at least 2 to 8 groups of eight consecutive Lys residues, Argresidues or a combination thereof, wherein each group of eightconsecutive lysine, arg or lys/arg residues is separated from adjacentgroups by 0-20 amino acid residues. More preferably, the nucleic acidbinding region of the single-chain antigen-binding polypeptide comprisesat least 2 to 6 groups of eight consecutive Lys residues, Arg residuesor a combination thereof, wherein each group of eight consecutivelysine, arg or lys/arg residues is separated from adjacent groups by0-20 amino acid residues. Still more preferably, the nucleic acidbinding region of the single-chain antigen-binding polypeptide comprisesat least 2 to 4 groups of eight consecutive Lys residues, Arg residuesor a combination thereof, wherein each group of eight consecutivelysine, arg or lys/arg residues is separated from adjacent groups by0-20 amino acid residues. More preferably, the nucleic acid bindingregion of the single-chain antigen-binding polypeptide comprises atleast 2 to 3 groups of eight consecutive Lys residues, Arg residues or acombination thereof, wherein each group of eight consecutive lysine, argor lys/arg residues is separated from adjacent groups by 0-20 amino acidresidues. Still more preferably, the nucleic acid binding region of thesingle-chain antigen-binding polypeptide has at least 2 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive lysine, arg or lys/arg residues isseparated from adjacent groups by 0-20 amino acid residues.

The nucleic acid binding regions of the single-chain antigen-bindingpolypeptide of the present invention can be represented by the followingformulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8), wherein K islysine, m is an integer between 1 and 7 and n is an integer between 0and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), wherein R isArginine, m is an integer between 1 and 7 and n is an integer between 0and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), wherein K islysine R is arginine such that the K and R residues either alternate orare in random order, m is an integer between 1 and 7 and n is an integerbetween 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQ ID NO:11),wherein K is lysine, R is arginine, m is an integer between 1 and 7 andn is an integer between 0 and 20.

Preferably, the nucleic acid binding regions of the single-chainantigen-binding polypeptide of the present invention can be representedby the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8),wherein K is lysine, m is an integer between 1 and 3 and n is an integerbetween 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), whereinR is Arginine, m is an integer between 1 and 5 and n is an integerbetween 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10) whereinK is lysine R is arginine such that the K and R residues eitheralternate or are in random order, m is an integer between 1 and 5 and nis an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQID NO:11), wherein K is lysine, R is arginine, m is an integer between 1and 5 and n is an integer between 0 and 20.

More preferably, the nucleic acid binding regions of the single-chainantigen-binding polypeptide of the present invention can be representedby the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8),wherein K is lysine, m is an integer between 1 and 3 and n is an integerbetween 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), whereinR is Arginine, m is an integer between 1 and 3 and n is an integerbetween 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), whereinK is lysine R is arginine such that the K and R residues eitheralternate or are in random order, m is an integer between 1 and 3 and nis an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQID NO:11), wherein K is lysine, R is arginine, m is an integer between 1and 3 and n is an integer between 0 and 20.

Still more preferably, the nucleic acid binding regions of thesingle-chain antigen-binding polypeptide of the present invention can berepresented by the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK)(SEQ ID NO:8), wherein K is lysine, m is an integer between 1 and 2 andn is an integer between 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR),wherein R is Arginine, m is an integer between 1 and 2 and n is aninteger between 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10),wherein K is lysine R is arginine such that the K and R residues eitheralternate or are in random order, m is an integer between 1 and 2 and nis an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQID NO:11), wherein K is lysine, R is arginine, m is an integer between 1and 2 and n is an integer between 0 and 20. More preferably, the DNAbinding regions of the single-chain antigen-binding polypeptide of thepresent invention can be represented by the following formulas: 1)(KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8), wherein K is lysine, m is 1and n is an integer between 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR)(SEQ ID NO:9), wherein R is Arginine, m is 1 and n is an integer between0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), wherein K islysine R is arginine such that the K and R residues either alternate orare in random order, m is 1 and n is an integer between 0 and 20; and 4)(RRRRRRRR)m ()on (KKKKKKKK) (SEQ ID NO:11), wherein K is lysine, R isarginine, m is 1 and n is an integer between 0 and 20.

Even more preferably, the single-chain antigen-binding polypeptide hasthe basic amino acid residue rich region, oligo-lysine residues,oligo-arginine residues or combination thereof, configured such that thenumber of groups of Lys and or Arg residues is no higher than that whichwould result in an unstable polynucleotide encoding the single-chainantigen-binding polypeptide or significantly reduce the efficiency oftranslation of the polynucleotide encoding the single-chainantigen-binding polypeptide or interfere with antigen binding.

The preferred ratio of single-chain antigen-binding polypeptide tonucleic acid are as follows: 10,000:1, 2000:1, 1000:1, 500:1, 250:1 or100:1 (molar ratio of sFv:DNA). Of course, these ranges are exemplaryonly and one of skill in the art could readily optimize the ratios foroptimal binding and transfection results for any particular cell ortissue type.

The optimal conditions for complexing the nucleic acids with the sFv ofthe present invention is as follows. Preferably, the complexing isperformed in a complexing buffer containing 0-100 mM Tris-HCl (or anyequivalent buffer), 5-500 mM NaCl, pH 6-9. More preferably, thecomplexing is performed in a buffer containing 10 mM Tris-HCl, 150 mMNaCl, pH 7.5. The time and temperature for complexing the nucleic acidswith the sFv in the foregoing complexing buffer are 1 to 120 minutes at4-40° C. The preferred time and temperature for complexing the nucleicacids with the sFv in the complexing buffer are 15 minutes at roomtemperature or about 22° C.

Chen et al., described the production and use of a fusion proteinconsisting of an antibody Fab fragment and a DNA binding moiety,protamine, to deliver toxin-expressing plasmid DNA into HIV infectedcells by receptor mediated endocytotsis. S-Y Chen et al., (1995) GeneTherapy 2: 116-123. The present invention, however, has advantages overthe Fab-protamine fusion peptide for delivering DNA into cells asdisclosed by Chen et al. This is because, protamine or poly lysinedomains (of a 100 lysine residues or more, i.e., “100K”) are very tightbinders of DNA compared to the “16K” tail of the present invention. Thepresent inventors, however, have discovered that the oligo lysineconfiguration of the present invention having 16 lysines (“16K”) placedwithin a short C-terminal extension from the sFv behaves as a minimalnucleic acid-binding domain. (See, for example, FIGS. 2A, 2B and 3 andExamples 1 and 3). This is because the natural DNA binding protein likeprotamine interacts with DNA presumably by electrostatic interactions,hydrogen bonding, hydrophobic bonds, Van der Waals bonds, and overallshape complementarity. However, in contrast to most natural DNA bindingproteins, the sFv containing a basic amino acid rich region according tothe present invention is proposed to bind and complex with nucleic acidsessentially through electrostatic interactions and interpolyelectrolytecomplex chemistry.

Moreover, Pardridge et al (J. Pharma. and Experimetal Therapeutics286:548-554 (1998)) have shown that “cationization” promotes endocytosisof Mabs. Thus, the sFv having a basic amino acid rich region accordingto the present invention can be more readily taken up by endocytosis duean increased positive charge of the sFv. An increase in endocytosis isexpected to result in increased transfection efficiencies and expressionof the nucleic acids that are complexed with the sFv of the presentinvention.

The use of sFv fused to a minimal nucleic acid-binding domain can alsohave production advantages. Although, proteins such as protamine orpolylysine having 100 or more lysines can be more effective atcondensing DNA, the expected reduction in affinity of the 16K tail forDNA, relative to protamine, will have the advantage of releasing thenucleic acid more efficiently from the sFv once targeting has beenachieved, thereby allowing this nucleic acid to be expressed by thecell. Thus, the 16K tailed sFv of the present can be a more effectivenucleic acid delivery vehicle than the Fab-protamine or Fab-polylysine(100K) synthetic polypeptides disclosed in the art. The oligo-lysine oroligo-arginine tail strategy of the present invention can be amenable toPEGylation as discussed, supra, which results in a DNA delivery carrierwith reduced immunogenicity and increased half-life.

As shown in Examples 5 and 6, below, the 16K sFv of the presentinvention can be employed as a targeting molecule to enhancetransfection of specific cells in culture. The demonstration of DNAdelivery to cultured cells by in situ immunochemistry shows that the SCAmolecule of the present invention can accomplish specific targeting,even for targets that are non-internalizing. In addition, transfectionwas also shown to be markedly enhanced by the oligo-lysine sFv of thepresent invention. Since the oligo-lysine sFv of the present inventionhas demonstrated to be successful in transfecting targets that are noninternalizing, it is anticipated that the enhanced specific transfectionof an internalizing target should also be achievable.

The nucleic acid used in the present invention can have a therapeuticeffect on the target cell, the effect selected from, but not limited to,correcting a defective gene or protein, a drug action, a toxic effect, agrowth stimulating effect, a growth inhibiting effect, a metaboliceffect, a catabolic affect, an anabolic effect, an antiviral effect, anantibacterial effect, a hormonal effect, a neurohumoral effect, a celldifferentiation stimulatory effect, a cell differentiation inhibitoryeffect, a neuromodulatory effect, an antineoplastic effect, ananti-tumor effect, an insulin stimulating or inhibiting effect, a bonemarrow stimulating effect, a pluripotent stem cell stimulating effect,an immune system stimulating effect, and any other known therapeuticeffects that can be provided by a therapeutic agent delivered to a cellvia a delivery system according to the present invention.

The sFv conjugate of the present invention can be used for protection,suppression or treatment of infection or disease. By the term“protection” from infection or disease as used herein is intended“prevention,” “suppression” or “treatment.” “Prevention” involvesadministration of a sFv conjugate prior to the induction of the disease.“Suppression” involves administration of the composition prior to theclinical appearance of the disease.

“Treatment” involves administration of the protective composition afterthe appearance of the disease. It will be understood that in human andveterinary medicine, it is not always possible to distinguish between“preventing” and “suppressing” since the ultimate inductive event orevents can be unknown, latent, or the patient is not determined untilwell after the occurrence of the event or events. Therefore, it iscommon to use the term “prophylaxis” as distinct from “treatment” toencompass both “preventing” and “suppressing” as defined herein. Theterm “protection,” as used herein, is meant to include “prophylaxis.”

Further, essentially all of the uses for which monoclonal or polyclonalantibodies, or fragments thereof, have been envisioned by the prior art,can be addressed by the oligo-lysine tailed sFv proteins of the presentinvention. See, e.g., Kohler et al., Nature 256:495 (1975); Kohler etal, Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292(1976); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681, Elsevier, N (1981); Sambrook et al., MolecularCloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory(1989).

The gene delivery system of the present invention can be used for anyhost. Preferably, the host will be a mammal. Preferred mammals includeprimates such as humans and chimpanzees, domestic animals such ashorses, cows, pigs, dogs, and cats. More preferably, the host animal isa primate or domestic animal. Still more preferably, the host animal isa primate such as a human.

Because humans are the desired hosts for in vivo delivery, certain testmodels have been developed and accepted by the field to determine theefficacy and utility of a delivery system. This involves in vitrotesting, ex vivo testing and use of marker genes. Thus, thesusceptibility of a cell to gene delivery by the method of the presentinvention can be determined by assays for a reporter gene. A marker genesuch as that encoding β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), etc. is used for convenience to determine whether aprotein can be expressed in a particular recombinant construct deliveredby the present method. In addition, the quantity and duration ofexpression can be assayed. The use of, for example neomycin resistance,to determine the efficacy of gene delivery has been described in humantesting with the desired gene. Thus, the skilled artisan, based on thisdisclosure can readily determine the efficacy of delivery of aparticular vector construct in a particular target tissue and host usingthe method of the present invention.

The genetic material (nucleic acids) that is delivered to the targetcell using the method of the present invention can be genes, forexample, those that encode a variety of proteins including anticancerand antiviral agents. Such genes include those encoding varioushormones, growth factors, enzymes, cytokines, receptors, MHC moleculesand the like. The term “genes” includes nucleic acid sequences bothexogenous and endogenous to cells into which the vector containing thegene of interest can be introduced.

Of particular interest for use in gene delivery are those genes encodingpolypeptides either absent, produced in diminished quantities orproduced in a mutant form in individuals suffering from a geneticdisease. Such genetic diseases include retinoblastoma, Wilms tumor,adenosine deaminase deficiency (ADA), thalassemias, cystic fibrosis,Sickle cell disease, Huntington's disease, Duchenne's musculardystrophy, Phenylketonuria, Lesch-Nyhan syndrome, Gaucher's disease,Tay-Sach's disease, and the like.

Additionally, it is of interest to use genes encoding tumor suppressorgenes (e.g., retinoblastoma gene), TNF, TGF-β, TGF-α, hemoglobin,interleukins, GM-CSF, G-CSF, M-CSF, human growth hormone, co-stimulatoryfactor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, EPO,β-globin and the like, as well as biologically active muteins of theseproteins. Genes for delivery to target cells can be from a variety ofspecies; however, preferred species sources for genes of interest arethose species into which the gene of interest is to be inserted usingthe method of the present invention.

The gene can further encode a product that regulates expression ofanother gene product or blocks one or more steps in a biologicalpathway, such as the sepsis pathway. In addition, the gene can encode atoxin fused to a polypeptide, e.g., a receptor ligand or an antibodythat directs the toxin to a target such as a tumor cell or a virus.Similarly, the gene can encode a protein that provides a therapeuticeffect to a diseased tissue or organ.

Basic techniques for operably inserting genes into expression vectorsare known to those skilled in the art See, Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989); Ausubel etal. (eds.), CURRENTPROTOCOLSIN MOLECULAR BIOLOGY, John Wiley and Sons (1987), both incorporatedherein by reference.

Several possible vector systems are available for the expression of thegene in the mammalian cell that has been transformed according to themethod of the present invention expression. These vector systems arewell known to those skilled in the art. For example, one class ofvectors utilize DNA elements which provide autonomously replicatingextra-chromosomal plasmids, derived from animal viruses such as bovinepapilloma virus, polyoma virus, adenovirus, or SV40 virus. A secondclass of vectors relies upon the integration of the desired genesequences into the host chromosome. Cells which have stably integratedthe introduced DNA into their chromosomes can be selected by alsointroducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker can provide forprototrophy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or resistance to heavy metals, such as copper or the like.The selectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements can also be needed for optimalsynthesis of mRNA. These elements can include splice signals, as well astranscription promoters, enhancers, and termination signals. The cDNAexpression vectors incorporating such elements include those describedby Okayama, H., Mol. Cell. Biol. 3:280(1983), and others.

Single Chain Polypeptides

The invention relates to the discovery that single-chain antigen-bindingproteins (“SCA”) or single-chain variable fragments of antibodies(“sFv”) having basic amino acid tails, have significant utility beyondthat of the non basic amino acid tailed single-chain antigen-bindingproteins. In addition to maintaining an antigen binding site, this SCAprotein has a basic amino acid region, such as oligo-lysine or oligoarginine or a combination thereof, according to the present invention asdisclosed, supra, at the C-terminus or N-terminus which is capable ofnon-specific nucleic acid binding thus enabling the basic amino acidtailed SCA polypeptide to act as a carrier to deliver nucleic acid tocells. Accordingly, the invention is directed to monovalent andmultivalent SCA proteins having an oligo-lysine tail, compositions ofmonovalent and multivalent basic amino acid tailed SCA proteins, methodsof making and purifying monovalent and multivalent basic amino acidtailed SCA proteins, and uses for the basic amino acid tailed SCAproteins. The invention is also directed to SCA proteins containingbasic amino acid regions having a diagnostic or therapeutic agent boundto the basic amino acid linked polypeptide.

The terms “single-chain antigen-binding molecule” (SCA) or “single-chainFv” (sFv) are used interchangeably. They are structurally defined ascomprising the binding portion of a first polypeptide from the variableregion of an antibody V_(L) (or V_(H)), associated with the bindingportion of a second polypeptide from the variable region of an antibodyV_(H) (or V_(L)), the two polypeptides being joined by a peptide linkerlinking the first and second polypeptides into a single polypeptidechain, such that the first polypeptide is N-terminal to the linker andsecond polypeptide is C-terminal to the first polypeptide and linker.The single polypeptide chain thus comprises a pair of variable regionsconnected by a polypeptide linker. The regions can associate to form afunctional antigen-binding site, as in the case wherein the regionscomprise a light-chain and a heavy-chain variable region pair withappropriately paired complementarity determining regions (CDRs). In thiscase, the single-chain protein is referred to as a “single-chainantigen-binding protein” or “single-chain antigen-binding molecule.”

Single-chain Fvs can and have been constructed in several ways. EitherV_(L) is the N-terminal domain followed by the linker and V_(H) (a V_(L)-Linker-V_(H) construction) or V_(H) is the N-terminal domain followedby the linker and V_(L) (V_(H) -Linker-V_(L) construction). Thepreferred embodiment contains V_(L) in the N-terminal domain (see,Anand, N. N., et al., J. Biol. Chem. 266:21874-21879 (1991)).Alternatively, multiple linkers have also been used. Several types ofsFv proteins have been successfully constructed and purified, and haveshown binding affinities and specificities similar to the antibodiesfrom which they were derived.

A description of the theory and production of single-chainantigen-binding proteins is found in Ladner et al., U.S. Pat. Nos.4,946,778, 5,260,203, 5,455,030 and 5,518,889, and in Huston et al.,U.S. Pat. No. 5,091,513 (“biosynthetic antibody binding sites” (BABS)),all incorporated herein by reference. The single-chain antigen-bindingproteins produced under the process recited in the above patents havebinding specificity and affinity substantially similar to that of thecorresponding Fab fragment.

Typically, the Fv domains have been selected from the group ofmonoclonal antibodies known by their abbreviations in the literature as26-10, MOPC 315, 741F8, 520C9, McPC 603, D1.3, murine phOx, human phOx,RFL3.8 sTCR, 1A6, Se155-4, 18-2-3, 4-4-20, 7A4-1, B6.2, CC49, 3C2, 2c,MA-15C5/K₁₂G₀, Ox, etc. (see, Huston, J. S. et al, Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988); Huston, J. S. et al, SIM News 38(4)(Supp):11 (1988); McCartney, J. et al., ICSU Short Reports 10:114(1990); McCartney, J. E. et al., unpublished results (1990); Nedelman,M. A. et al., J. Nuclear Med. 32 (Supp.):1005 (1991); Huston, J. S. etal., In: Molecular Design and Modeling: Concepts and Applications, PartB, edited by J. J. Langone, Methods in Enzymology 203:46-88 (1991);Huston, J. S. et al., In: Advances in the Applications of MonoclonalAntibodies in Clinical Oncology, Epenetos, A. A. (Ed.), London, Chapman& Hall (1993); Bird, R. E. et al., Science 242:423-426 (1988); Bedzyk,W. D. et al., J. Biol. Chem. 265:18615-18620 (1990); Colcher, D. et al.,J. Nat. Cancer Inst. 82:1191-1197 (1990); Gibbs, R. A. et al., Proc.Natl. Acad. Sci. USA 88:4001-4004 (1991); Milenic, D. E. et al., CancerResearch 51:6363-6371 (1991); Pantoliano, M. W. et al., Biochemistry30:10117-10125 (1991); Chaudhary, V. K. et al., Nature 339:394-397(1989); Chaudhary, V. K. et al., Proc. Natl. Acad Sci. USA 87:1066-1070(1990); Batra, J. K. et al., Biochem. Biophys. Res. Comm. 171:1-6(1990); Batra, J. K. et al, J. Biol. Chem. 265:15198-15202 (1990);Chaudhary, V. K. et al., Proc. Natl. Acad. Sci USA 87:9491-9494 (1990);Batra, J. K. et al., Mol. Cell. Biol. 11:2200-2205 (1991); Brinkmann, U.et al., Proc. Natl. Acad Sci. USA 88:8616-8620 (1991); Seetharam, S. etal., J. Biol. Chem. 266:17376-17381 (1991); Brinkmann, U. et al., Proc.Natl. Acad Sci. USA 89:3075-3079 (1992); Glockshuber, R. et al.,Biochemistry 29:1362-1367 (1990); Skerra, A. et al., Bio/Technol.9:273-278 (1991); Pack, P. et al., Biochemistry 31:1579-1534 (1992);Clackson, T. et al., Nature 352:624-628 (1991);Marks, J. D. et al., J.Mol. Biol. 222:581-597 (1991); Iverson, B. L. et al., Science249:659-662 (1990); Roberts, V. A. et al., Proc. Natl. Acad. Sci. USA87:6654-6658 (1990); Condra, J. H. et al., J. Biol. Chem. 265:2292-2295(1990); Laroche, Y. et al., J. Biol. Chem. 266:16343-16349 (1991);Holvoet, P. et al., J. Biol. Chem. 266:19717-19724 (1991); Anand, N. N.et al., J. Biol. Chem. 266:21874-21879 (1991); Fuchs, P. et al.,Bio/Technol. 9:1369-1372 (1991); Breitling, F. et al., Gene 104:104-153(1991); Seehaus, T. et al., Gene 114:235-237 (1992); Takkinen, K. etal., Protein Engng. 4:837-841 (1991); Dreher, M. L. et a., J. Immunol.Methods 139:197-205 (1991); Mottez, E. et al., Eur. J. Immunol.21:467-471 (1991); Traunecker, A. et al., Proc. Natl. Acad. Sci. USA88:8646-8650(1991); Traunecker, A. et al., EMBO J. 10:3655-3659 (1991);Hoo, W. F. S. et al., Proc. Natl. Acad Sci. USA 89:4759-4763 (1993)).

Linkers of the invention used to construct sFv polypeptides are designedto span the C-terminus of V_(L) (or neighboring site thereof) and theN-terminus of V_(H) (or neighboring site thereof). The preferred lengthof the peptide linker should be from 2 to about 50 amino acids. In eachparticular case, the preferred length will depend upon the nature of thepolypeptides to be linked and the desired activity of the linked fusionpolypeptide resulting from the linkage. Generally, the linker should belong enough to allow the resulting linked fusion polypeptide to properlyfold into a conformation providing the desired biological activity.Where conformational information is available, as is the case with sFvpolypeptides discussed below, the appropriate linker length can beestimated by consideration of the 3-dimensional conformation of thesubstituent polypeptides and the desired conformation of the resultinglinked fusion polypeptide. Where such information is not available, theappropriate linker length can be empirically determined by testing aseries of linked fusion polypeptides with linkers of varying lengths forthe desired biological activity. Such linkers are described in detail inWO 94/12520, incorporated herein by reference.

Preferred linkers used to construct sFv polypeptides have between 10 and30 amino acid residues. The linkers are designed to be flexible, and itis recommended that an underlying sequence of alternating Gly and Serresidues be used. To enhance the solubility of the linker and itsassociated single chain Fv protein, three charged residues can beincluded, two positively charged lysine residues (K) and one negativelycharged glutamic acid residue (E). Preferably, one of the lysineresidues is placed close to the N-terminus of V_(H), to replace thepositive charge lost when forming the peptide bond of the linker and theV_(H). Such linkers are described in detail in U.S. patent applicationSer. No. 08/224,591, now U.S. Pat. No. 5,856,456, filed Apr. 7, 1994,incorporated herein by reference. See also, Whitlow, M., et al., ProteinEngng. 7:1017-1026 (1994). It should also be noted that a basic aminoacid region having lysine and arginine residues could also be used inthe linker for the sFv polypeptides of the present invention.

For multivalent sFvs, the association of two or more sFvs is requiredfor their formation. Although, multivalent sFvs can be produced fromsFvs with linkers as long as 25 residues, they tend to be unstable.Holliger, P., et al., Proc. Natl. Acad Sci. USA 90:6444-6448 (1993),have recently demonstrated that linkers 0 to 15 residues in lengthfacilitate the formation of divalent Fvs. See, Whitlow, M., et al.,Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R., Nature Biotech.15:125-126 (1997). Such multivalent sFvs are described in detail in WO93/11161, herein incorporated by reference.

Furthermore, single-chain and multivalent immunoeffector antigen-bindingfusion proteins have also been designed and constructed. Suchsingle-chain and multivalent immnunoeffector antigen-binding fusionproteins provide the binding capability of the antigen binding proteincombined with the immunoeffector or cytolytic function fusion partner,such as TNF, PLAP, IL-2, GM-CSF and the like. Such single-chain andmultivalent immunoeffector antigen-binding fusion proteins are describedin detail in U.S. Pat. No. 5,763,733, incorporated by reference.

The object of the present invention is to produce an sFv having anucleic acid binding region comprising basic amino acid residues. Thenucleic acid binding region can comprise a sequence that is rich inbasic amino acids such as lysine, arginine and combinations thereof.This region will contain enough basic amino acids to obtain therequisite overall positive charge on the sFv for nucleic acid binding.These nucleic acid binding regions can be at the C-terminal region,N-terminal region or both of the sFv. The nucleic acid binding regionscan be generated by mutating one or a plurality of amino acid residue(s)or by adding a block of basic amino acid residues to the C-terminalregion, N-terminal region or both of the sFv. The sFv can have a regionrich in basic amino acids, an oligo-Lys, oligo-Arg or combinationthereof as a tail such that the basic amino acid rich region, oligo-Lysor oligo-Arg residues are sufficient to bind nucleic acids and thepolypeptide binds an antigen (i.e., the polypeptide's ability to bind anantigen is not disrupted).

Preferably, the nucleic acid binding region of the single-chainantigen-binding polypeptide comprises at least 2 to 8 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive lysine, arg or lys/arg residues isseparated from adjacent groups by 0-20 amino acid residues. Morepreferably, the nucleic acid binding region of the single-chainantigen-binding polypeptide comprises at least 2 to 6 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive lysine, arg or lys/arg residues isseparated from adjacent groups by 0-20 amino acid residues. Still morepreferably, the nucleic acid binding region of the single-chainantigen-binding polypeptide comprises at least 2 to 4 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive lysine, arg or lys/arg residues isseparated from adjacent groups by 0-20 amino acid residues. Morepreferably, the nucleic acid binding region of the single-chainantigen-binding polypeptide comprises at least 2 to 3 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive lysine, arg or lys/arg residues isseparated from adjacent groups by 0-20 amino acid residues. Still morepreferably, the nucleic acid binding region of the single-chainantigen-binding polypeptide has at least 2 groups of eight consecutiveLys residues, Arg residues or a combination thereof, wherein each groupof eight consecutive lysine, arg or lys/arg residues is separated fromadjacent groups by 0-20 amino acid residues.

Alternatively, the nucleic acid binding regions of the single-chainantigen-binding polypeptide of the present invention can be representedby the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8),wherein K is lysine, m is an integer between 1 and 7 and n is an integerbetween 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), whereinR is Arginine, m is an integer between 1 and 7 and n is an integerbetween 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), whereinK is lysine R is arginine such that the K and R residues eitheralternate or are in random order, m is an integer between 1 and 7 and nis an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQID NO:11), wherein K is lysine, R is arginine, m is an integer between 1and 7 and n is an integer between 0 and 20.

Preferably, the DNA binding regions of the single-chain antigen-bindingpolypeptide of the present invention can be represented by the followingformulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8), wherein K islysine, m is an integer between 1 and 5 and n is an integer between 0and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), wherein R isArginine, m is an integer between 1 and 5 and n is an integer between 0and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), wherein K islysine R is arginine such that the K and R residues either alternate orare in random order, m is an integer between 1 and 5 and n is an integerbetween 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQ ID NO:11),wherein K is lysine, R is arginine, m is an integer between 1 and 5 andn is an integer between 0 and 20.

More preferably, the nucleic acid binding regions of the single-chainantigen-binding polypeptide of the present invention can be representedby the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8),wherein K is lysine, m is an integer between 1 and 3 and n is an integerbetween 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), whereinR is Arginine, m is an integer between 1 and 3 and n is an integerbetween 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ ID NO:10), whereinK is lysine R is arginine such that the K and R residues eitheralternate or are in random order, m is an integer between 1 and 3 and nis an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQID NO:11), wherein K is lysine, R is arginine, m is an integer between 1and 3 and n is an integer between 0 and 20.

Still more preferably, the nucleic acid binding regions of thesingle-chain antigen-binding polypeptide of the present invention can berepresented by the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK)(SEQ ID NO:8), wherein K is lysine, m is an integer between 1 and 2 andn is an integer between 0 and 20; 2) (RRRRRRRR)m (X)n (RRRRRRRR) (SEQ IDNO:9), wherein R is Arginine, m is an integer between 1 and 2 and n isan integer between 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK) (SEQ IDNO:10), wherein K is lysine R is arginine such that the K and R residueseither alternate or are in random order, m is an integer between 1 and 2and n is an integer between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK)(SEQ ID NO:11), wherein K is lysine, R is arginine, m is an integerbetween 1 and 2 and n is an integer between 0 and 20.

More preferably, the nucleic acid binding regions of the single-chainantigen-binding polypeptide of the present invention can be representedby the following formulas: 1) (KKKKKKKK)m (X)n (KKKKKKKK) (SEQ ID NO:8),wherein K is lysine, m is 1 and n is an integer between 0 and 20; 2)(RRRRRRRR)m (X)n (RRRRRRRR) (SEQ ID NO:9), wherein R is Arginine, m is 1and n is an integer between 0 and 20; 3) (RKRKRKRK)m (X)n (RKRKRKRK)(SEQ Id NO:10), wherein K is lysine R is arginine such that the K and Rresidues either alternate or are in random order, m is 1 and n is aninteger between 0 and 20; and 4) (RRRRRRRR)m (X)n (KKKKKKKK) (SEQ IDNO:11), wherein K is lysine, R is arginine, m is 1 and n is an integerbetween 0 and 20.

Even more preferably, the single-chain antigen-binding polypeptide hasthe region rich in basic amino acid residues, oligo lysine residues,oligo arginine residues or combination thereof, configured such that thenumber of groups of consecutive Lys and or Arg residues is no higherthan that which would result in an unstable polynucleotide encoding thesingle-chain antigen-binding polypeptide or significantly reduce theefficiency of translation of the polynucleotide encoding thesingle-chain antigen-binding polypeptide.

These novel sFv proteins can be conjugated to activated polyethyleneglycol (PEG) such that the PEG modification occurs preferentially atspecifically engineered sites. See, U.S. application Ser. No.09/069,842,filed on Apr. 30,1998.

A further object of the invention is to produce monovalent andmultivalent sFvs having the oligo lysine tails of the present invention.For multivalent sFv, the association of two or more sFvs is required fortheir formation. For example, multivalent sFvs can be generated bychemically crosslinking two sFvs with C-terminal cysteine residues(Cumber et al., J. Immunol. 149:120-126 (1992)) and by linking two sFvswith a third polypeptide linker to form a dimeric Fv (George et aL, J.Cell. Biochem. 15E:127 (1991)). Details for producing multivalent sFvsby aggregation are described in Whitlow, M., et al., Protein Engng.7:1017-1026 (1994). Multivalent antigen-binding fusion proteins of theinvention can be made by any process, but preferably according to theprocess for making multivalent antigen-binding proteins set forth in WO93/11161, incorporated herein by reference.

Synthesis of the Minimal Nucleic Acid Binding Regions

In the present invention, a region rich in basic amino acid residues,oligo-Lys, oligo-Arg or oligo-Lys/Arg nucleic acid binding region canoccur in the C-terminus or N-terminus of the sFv polypeptide.Preferably, the nucleic acid binding region will occur in the C-terminusof the sFv polypeptide. The site at the C-terminus was chosen to be asfar from the antigen binding residues of the polypeptide as possible soas to prevent disruption of the antigen-binding site.

Site-directed mutagenesis is used to change the native protein sequenceof the single-chain antigen-binding protein to one that incorporates theregions rich in Lys, Arg, oligo-Lys, oligo-Arg or oligo-Lys/Argresidues. The mutant protein gene is placed in an expression system,such as bacterial cells, yeast or other fungal cells, insect cells ormammalian cells. The mutant protein can be purified by standardpurification methods.

Oligonucleotide-directed mutagenesis methods for generating the minimalbasic amino acid nucleic acid binding regions or the present inventionand related techniques for mutagenesis of cloned DNA are well known inthe art. See, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989);Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons (1987), both incorporated herein by reference. Apreferred oligonucleotide-directed mutagenesis method for the presentinvention is according to Ho et al., Gene 77:51-59 (1989), incorporatedherein by reference.

Hosts and Vectors for the Preparation of the sFv Polypeptides

After mutating the nucleotide sequence of the sFv, the mutated DNA canbe inserted into a cloning vector for further analysis, such as forconfirmation of the DNA sequence. To express the polypeptide encoded bythe mutated DNA sequence, the DNA sequence is operably linked toregulatory sequences controlling transcriptional expression andintroduced into either a prokaryotic or eukaryotic host cell.

Although sFvs are typically produced by prokaryotic host cells,eukaryotic cells can also be used as host cells. Preferred host cellsinclude E. coli, yeast or other fungal cells, insect cells or mammaliancells. Standard protein purification methods can be used to purify thesemutant proteins. Only minor modification to the native protein'spurification scheme can be required.

Also provided by the invention are DNA molecules such as purifiedgenetic sequences or plasmids or vectors encoding the sFv of theinvention that have engineered region(s) containing high content ofbasic amino acids, oligo-Lys, oligo-Arg or oligo-Lys/Arg residuescapable of non-specific nucleic acid binding. The DNA sequence for thesFv polypeptide can be chosen so as to optimize production in organismssuch as prokaryotes, yeast or other fungal cells, insect cells ormammalian cells.

The DNA molecule encoding an sFv having a region rich in basic aminoacid residues, oligo-Lys, oligo-Arg, or oligo-Lys/Arg residues whichcomprise the minimal DNA binding region can be operably linked into anexpression vector and introduced into a host cell to enable theexpression of the engineered sFv protein by that cell. A DNA sequenceencoding an sFv having a region rich in basic amino acid residues,oligo-Lys, oligo-Arg, or oligo-LysArg regions can be recombined withvector DNA in accordance with conventional techniques. Recombinant hostsas well as methods of using them to produce single chain proteins of theinvention are also provided herein.

The expression of such sFv proteins of the invention can be accomplishedin procaryotic cells. Preferred prokaryotic hosts include, but are notlimited to, bacteria such as Bacilli, Streptomyces and E. coli.

Eukaryotic hosts for cloning and expression of such sFv proteins of theinvention include plant cells, insect cells, yeast, fungi, and mammaliancells (such as, for example, human or primate cells) either in vivo, orin tissue culture. A preferred host for the invention is Pichiapastoris. As discussed in more detail below, the inventors havedemonstrated excellent yields of the sFv proteins having the regionsrich in basic amino acid residues according to the present inventionusing Pichia pastoris.

The appropriate DNA molecules, hosts, methods of production, isolationand purification of monovalent, multivalent and fusion forms ofproteins, especially sFv polypeptides, are thoroughly described in theprior art, such as, e.g., U.S. Pat. No. 4,946,778, which is fullyincorporated herein by reference.

The sFv encoding sequence having the minimal DNA binding regioncomprising oligo-Lys, oligo-Arg, or oligo-Lys/Arg residues and anoperably linked promoter can be introduced into a recipient prokaryoticor eukaryotic cell either as a non-replicating DNA (or RNA) molecule,which can either be a linear molecule or, more preferably, a closedcovalent circular molecule. Since such molecules are incapable ofautonomous replication, the expression of the desired sFv protein canoccur through the transient expression of the introduced sequence.Alternatively, permanent expression can occur through the integration ofthe introduced sFv sequence into the host chromosome.

In one embodiment, the sFv sequence can be integrated into the host cellchromosome. Cells which have stably integrated the introduced DNA intotheir chromosomes can be selected by also introducing one or moremarkers which allow for selection of host cells which contain the sFvsequence and marker. The marker can complement an auxotrophy in the host(such as his4, leu2, or ura3, which are common yeast auxotrophicmarkers), or can confer biocide resistance, e.g., antibiotics, orresistance to heavy metals, such as copper, or the like. The selectablemarker gene can either be directly linked to the sFv DNA sequence to beexpressed, or introduced into the same cell by co-transfection.

In another embodiment, the introduced sequence will be incorporated intoa plasmid vector capable of autonomous replication in the recipient hostcell. Any of a wide variety of vectors can be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector canbe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Any of a series of yeast vector systems can be utilized. Examples ofsuch expression vectors include the yeast 2-micron circle, theexpression plasmids YEP13, YCP and YRP, etc., or their derivatives. Suchplasmids are well known in the art (Botstein et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes can be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker can provide prototrophyto an auxotrophic host, biocide resistance, e.g., antibiotics, orresistance to heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements can also be needed for optimal synthesis of mRNA.These elements can include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,H., Mol. Cell. Biol. 3:280 (1983), and others.

Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Preferred vectors for expression in Pichia are pHIL-S 1(Invitrogen Corp.) and pIC9 (Invitrogen Corp.). Other suitable vectorswill be readily apparent to the skilled artisan.

Once the vector or DNA sequence containing the sFv constructs of thepresent invention has been prepared for expression, the DNA constructscan be introduced or transformed into an appropriate host. Varioustechniques can be employed, such as transformation, transfection,protoplast fusion, calcium phosphate precipitation, electroporation, orother conventional techniques. After the cells have been transformedwith the recombinant DNA (or RNA) molecule, the cells are grown in mediaand screened for appropriate activities. Expression of the sequenceresults in the production of the mutant sFv for use in the gene deliverymethod of the present invention.

Expression and Purification of sFv Proteins

The inventors have demonstrated excellent yields of the oligo-lysinetailed sFv of the present invention, particularly CC49-16K, secretedfrom Pichia pastoris. This provides a means of making enough of theDNA-binding sFv for commercial gene therapy applications. In addition,the inventors have discovered a novel purification procedure for theoligo-lysine tailed sFv which is ionically bound to the nucleic acidsfrom lysed cells in the fermentation broth. The oligo-lysine tailed sFvis initially found in the cell pellet fraction, but can be readilyreleased by salt treatment in excellent yield and purity.

The vectors pIC9 and pHIL-S1, and host strain GS 115 were obtained fromInvitrogen Corporation and all cloning and expression work was performedas described in the “Pichia Expression Kit Instruction, Manual” suppliedby Invitrogen. Clone number designations for CC49/218 sFv variants areas follows.

Clone # Plasmid # Vector C-terminal lysine # EN266(5) pEN262(5) pHIL-S1 8 K BN266(7) pEN262(7) pHIL-S1 16 K EN281(1) pEN278(1) pIC9  8 K EN282pEN278(5) pIC9 16 K

Expression levels were >20 mg/L of protein as estimated by SDS-PAGE andWestern analysis. The 8K (8 lysine tail) version and 16K (16 lysinetail) version of the sFv migrated on SDS-PAGE at positions approximately1.6 KD and 3.3 KD greater in mass in agreement with the predicted sizefrom their polypeptide sequences. The sFv proteins were all soluble inshake-flask experiments, but often were associated with the cell pelletin fermentation cultures. The sFv was dissociated from the pellet byhigh salt wash (1.5 M NaCl, 20 mM Tris-HCl, pH 8.0 at room temperaturefor 2 hours). Consequently, this provided a very good purification step.Fermentation cultures contain substantial amounts of lysed cells and the16K sFv variant proteins appear to bind to nucleic acids present in thefermentation medium. Significantly, the native sFv does not become cellassociated. The Coomassie Blue stained SDS-PAGE gel of FIG. 5, is anexample of the excellent expression of CC4916K 266(7) and the ability ofsalt treatment to solubilize and purify the sFv of the presentinvention.

Western analysis confirmed the major sFv molecules at about 26.5 Kd fornative sFv and about 30 Kd for CC49-16K sFv. This experiment wasperformed as follows: (1) 100 ml of expression medium of EN266(7) fromshake-flask culture was frozen and thawed, then centrifuged at 3,000rpm, room temperature (RT) for 30 min.; (2) EN266 cell pellet wasresuspended in 2 ml of 1.5 M NaCl, 20 mM Tris-HCl, pH 8.0, RT, 2 hrs.;(3) the sample was centrifuged as in (1) and the supernatant wasdialyzed against 0.15 M NaCl, 10 mM Tris-HCl, pH 8.0 at 4° C. overnight;(4) the protein content of the supernantant was quantitated at A280 tobe about 1.5 mg/ml; and (5) 30 μl of the supernatant were loaded onSDS-PAGE gels for Coomassie Blue staining and Western analysis.

The Western analysis was performed as follows: Immunoblotting proceduresfor transfer of proteins from gels to nitrocellulose membranes by thesemi-dry method were performed as described in Harlow, E., & Lane, D.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988). Blot development was also performedaccording to the procedures in this manual. Briefly, the blottedmembranes were blocked in 1% BSA blocking reagent in PBS at roomtemperature for 2 hr; washed 3× with PBS; and incubated with 3% BSA inPBS with a 1:1,000 dilution of rabbit anti-CC49/218 SCA antibody at 4°C. overnight. Next, a 3% BSA in PBS solution containing a 1:1000dilution of horseradish peroxidase conjugated goat anti-rabbit IgG wasused in a 1 hr incubation at room temperature. After washing with PBS,the membranes were developed with TNBM-500 (MOSS, Inc.) at roomtemperature for 1 min.

The purified sFvs of the present invention can be stored as a stabilizedprotein composition having increased frozen storage stability asdescribed in detail in U.S. Pat. No.5,656,730, incorporated herein byreference.

Administration

Administration of basic amino acid tailed sFv-nucleic acid conjugates ofthe invention for ex vivo and in vivo delivery of nucleic acids tomammalian cells will be by analogous methods to sFv where the diagnosticor therapeutic principle is directly linked to the sFv or a loadedcarrier is linked by random binding to amine or carboxyl groups on aminoacid residues of the sFv in a non-site-specific manner.

Conjugates of the present invention (immunoconjugates) can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, such as by admixture with a pharmaceutically acceptablecarrier vehicle. Suitable vehicles and their formulation are described,for example, in Remington's Pharmaceutical Sciences, 18th ed., Osol, A.,ed., Mack, Easton Pa. (1990). In order to form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain a therapeutically effective amount of theimmunoconjugate, either alone, or with a suitable amount of carriervehicle.

The immunoconjugate can be provided to a patient by means well known inthe art. Such means of introduction include subcutaneous means,intramuscular means, intravenous means, intra-arterial means, orparenteral means. Intravenous, intraarterial or intrapleuraladministration is normally used for lung, breast, and leukemic tumors.Intraperitoneal administration is advised for ovarian tumors.Intrathecal administration is advised for brain tumors and leukemia.Subcutaneous administration is advised for Hodgkin's disease, lymphomaand breast carcinoma. Catheter perfusion is useful for metastatic lung,breast or germ cell carcinomas of the liver. Intralesionaladministration is useful for lung and breast lesions.

For therapeutic or diagnostic applications, compositions according tothe invention can be administered parenterally in combination withconventional injectable liquid carriers such as sterile pyrogen-freewater, sterile peroxide-free ethyl oleate, dehydrated alcohol, orpropylene glycol. Conventional pharmaceutical adjuvants for injectionsolution such as stabilizing agent, solubilizing agents and buffers,such as ethanol, complex forming agents such as ethylene diaminetetraacetic acid, tartrate and citrate buffers, and high-molecularweight polymers such as polyethylene oxide for viscosity regulation canbe added. Such compositions can be injected intramuscularly,intraperitoneally, or intravenously.

Further non-limiting examples of carriers and diluents include albuminand/or other plasma protein components such as low density lipoproteins,high density lipoproteins and the lipids with which these serum proteinsare associated. These lipids include phosphatidyl choline, phosphatidylserine, phosphatidyl ethanolamine and neutral lipids such astriglycerides. Lipid carriers also include, without limitation,tocopherol.

A typical regimen for preventing, suppressing, or treating variouspathologies comprises administration of an effective amount of an sFvconjugate, administered over a period of one or several days, up to andincluding between one week and about 24 months.

It is understood that the dosage of the present invention administeredin vivo or in vitro will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. The ranges ofeffective doses provided below are not intended to limit the inventionand represent preferred dose ranges. However, the most preferred dosagewill be tailored to the individual subject, as is understood anddeterminable by one of skill in the art, without undue experimentation.See, e.g, Berkow et al, eds., Merck Manual, 16th edition, Merck and Co.,Rahway, N.J. (1992); Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston (1985), Katzung, Basic andClinical Phamacology, Appleton and Lange, Norwalk, Conn. (1992), whichreferences and references cited therein, are entirely incorporatedherein by reference.

The total dose required for each treatment can be administered bymultiple doses or in a single dose. Effective amounts of adiagnostic/pharmaceutical compound or composition of the presentinvention are from about 0.001 μg to about 100 mg/kg body weight,administered at intervals of 4-72 hours, for a period of 2 hours to 5years, or any range or value therein, such as 0.01-1.0, 1.0-10, 10-50and 50-100 mg/kg, at intervals of 1-4,6-12, 12-24 and 24-72 hours, for aperiod of 0.5, 1.0-2.0, 2.0-4.0 and 4.0-7.0 days, or 1, 1-2, 2-4, 4-52or more weeks, or 1, 2, 3-10, 10-20, 20-60 or more years, or any rangeor value therein.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which can containauxiliary agents or excipients which are known in the art. See, e.g.,Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which areentirely incorporated herein by reference, including all referencescited therein.

Pharmaceutical compositions comprising at least one type of sFvconjugate having a basic amino acid rich region according to theinvention, or, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types of sFv conjugates,of the present invention can be contained in an amount effective toachieve its intended purpose. In addition to at least one sFv conjugate,a pharmaceutical composition can contain suitable pharmaceuticallyacceptable carriers, such as excipients, carriers and/or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically.

Pharmaceutical compositions can also include suitable solutions foradministration intravenously, subcutaneously, dermally, orally,mucosally or rectally, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active component (i.e., theDNA binding sFv conjugate) together with the excipient. Pharmaceuticalcompositions for oral administration include tablets and capsules.Additional lipid and lipoprotein drug delivery systems that can beincluded herein are described more fully in Annals N.Y. Acad. Sci.507:775-88, 98-103, and 252-271, which disclosure is hereby incorporatedby reference.

For example, the sFvs of the present invention can be prepared as apharmaceutically acceptable, single-chain antigen-binding proteincomposition having increased frozen-storage stability, as described indetail in U.S. Pat. No. 5,656,730, incorporated by reference.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedfor the purpose of illustration and not intended to be limiting unlessotherwise specified.

EXAMPLES Example 1 Demonstration of DNA Binding

In order to demonstrate the DNA binding capabilities of the C-terminaloligo-lysine tailed SCAs the following experiment was performed. TheCC49/218 SCA and A33/218 SCA having a 16 lysine (“16K”) C-terminal tailand the CC49/218 SCA and A33/218 SCA having a 8 lysine (“8K”) C-terminaltail were expressed from Pichia.

Genetic construction of the sFv proteins having an oligo-lysineC-terminal tails was performed by first introducing a unique BstEIIrestriction site (GGTNACC) into VH codons including positions 108, 109and 110 (Kabat numbers) by standard site directed mutagenesis. Thismutation does not alter the encoded amino acids and is accomplished bysimply changing the position 108 codon from TCA (Ser) to TCG (Ser), asingle base change. The unique BstEII restriction site can be digestedwith the restriction enzyme BstEII and a synthetic linker having BstEIIcompatible overhangs is ligated into the site. The synthetic linker usedconsists of two complementary oligonucleotides:

5′ GTC ACC GTC TCC AAA AAG AAG AAA AAA AAG AAA AAG 3 (SEQ ID NO:12), and

5′ GT GAC CTT TTT CTT TTT TTT CTT CTT TTT GAA GAC G 3′ (SEQ ID NO:13).

This linker can be inserted as a single copy or as two or more tandemcopies due to the compatible overhangs. In the case of the 16 lysinetail sFv, two tandem copes of this linker are presented as confirmationby DNA sequencing of the genetic construction.

The proteins were assayed for DNA binding finction in a standard GelShift assay (Mistry et al. Biotechniques 22:718-729 (1997)). The A33/218SCA having a 16 lysine C-terminal tail (EN266 (3F)) was incubated withplasmid pFLAG-1(International Biotechnologies, Inc.) (0.5 μg) in DNAbinding buffer (0.01M Tris, pH8.0, 0.15M NaCl). The samples were thenelectrophoresed on the gel shown in FIG. 4. The results show that thisSCA protein had DNA binding capability. The CC49/218 SCA having a 16lysine C-terminal tail (EN278(5)) also bound DNA. The results in FIG. 4show that supercoiled DNA species (faster moving species) was moreeffectively complexed by the SCA molecules than the nicked linear DNAspecies and are consistent with the results shown by Mistry et al. TheCC49/218 SCA and A33/218 SCA having a 8 lysine C-terminal tail, however,did not show DNA binding capacity by this assay.

Example 2 Transfection of Mammalian Cells

In order to demonstrate the transfection of mammalian cells using theoligo-lysine single-chain antigen binding polypeptide of the presentinvention the following experiment can be performed.

CC49/218 sFv protein engineered to contain a 16 lysine tail (FIGS. 2Aand B) is expressed and secreted by Pichia pastoris strain EN266. Theprotein is purified by standard cation and anion exchange chromatographywell known to those skilled in the art. The protein is then concentratedby diafiltration. The sFv is incubated with reporter plasmid DNA, suchas one of the pRL vectors (Promega Corp.). The sFv and plasmid DNA areincubated for 10-60 minutes in buffer (0.01M Tris, pH 8.0,0.15M NaCl) inthe following concentration ratio: 0.5 μg of pRL vector and 10 μg of sFvpolypeptide. Controls for the transfection experiment are 1) CC49/218sFv lacking the 16 lysine tail incubated with pRL vector and 2) plasmidalone.

The sFv/plasmid complex is then incubated with cultured LS-174T cellswhich are resuspended in the buffer (0.01 M Tris, pH 8.0, 0.15 M NaCl),for 10-60 minutes. The cells are then centrifuged at 2,000 rpm for fiveminutes and washed once with incubation buffer. The cells are nextsuspended in electroporation buffer (1×HBS: 20 mM HEPES, pH 7.05, 137mMNaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose). One set of cells aresubjected to electroporation using BTX Electro Cell Manipulator 600System according to the manufacturer's instructions. Another cell ofcells are examined for the spontaneous uptake of the sFv/plasmid complexby omitting the electroporation step. The success of transfection of thecells by the reporter plasmid is quantitated by luciferase assaysperformed according to the protocol described by Promega Corp., inPromega Notes #57.

Example 3 Demonstration of DNA Binding

Additional Gel Shift assays demonstrating the DNA binding capacity ofCC49-16K (EN266(7)) and A33-16K were performed as follows. Experimentalconditions are as in Example 1 except as follows. CC49-16K (EN266(7))was purified by DEAE column chromatography and fraction 8 (OD280=3.6)was dialyzed versus 0.15 M NaCl, 10 mM Tris-HCl, pH 8.0 at 4° C.overnight. Aliquots of the samples (0-90 μl) were mixed with 1 μl ofplasmid Bluescript SK⁻ (3 ug/μl) and distilled water was added to afinal volume of 100 μl. The samples were incubated at RT for 1 hr.Twenty μl of each sample were loaded and run on a 1.2% agarose gel,100V, 2 hrs.

The A33-16K sample was incubated as in Example 1 except the plasmid usedwas pFLAG from IBI, Inc. (1 μg/μl) and incubation was done at RT for 1hr.

Example 4 ELISA Assay

An ELISA assay demonstrating retention of mucin-binding activity of theCC49-16K sFv EN266(7) shown in FIG. 6 was performed as follows. TheELISA was performed by 1:2 serial dilutions of the sFv samples.Immunoassay procedures were performed using modifications of protocolsfrom Harlow, E., & Lane, D., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). Directbinding assays were performed and a dose response curve was constructed.Bovine submaxillary mucin (250 ng per 100 μl well) antigen was used tocoat microtiter plate wells (MaxiSorp, Nunc, VWR Scientific, Boston,Mass.). The EN266(7) or purified CC49/218 SCA proteins were dilutedserially in PBS containing 1% BSA and incubated in the coated wells at22° C. for 1 hr. After the plate was washed three times with PBScontaining 0.05% Tween 20 (PBS-T), the bound SCA was detected by a 1 hrincubation with a secondary antibody (rabbit anti-CC49/218, 1:2000dilution, 37° C.), followed by three PBS-T washes, and a 1 hr incubationat 37° C. with horseradish peroxidase conjugated goat anti-rabbit IgGantibody (1:2000 dilution). Plates were washed 3 times with PBS-T andwere read at 540 nm following, addition of 100 μl of3′,3′,5′,5′-tetramethylbenzidine (TMB).

EN234 is native CC49/218 sFv produced from Pichia. GX9251 is nativeCC49/218 sFv produced from E coli. The BSA control is not plotted on thegraph. These results indicate that antigen-binding activity of the sFvis not substantially altered in the sFv-16K variant protein.

In the next experiments, these oligo-lysine tailed sFv proteins wereshown to be capable of gene delivery in vitro. First, these resultsdemonstrate that CC49-16K sFv which is complexed with plasmid DNAadheres to TAG-72 antigen bearing LS174-T cells. Second, these resultsdemonstrate that the CC49-16K sFv/plasmid complex can markedly enhancethe LipofectAmine transfection of DNA into the LS174-T cells.

Example 5 Targeted DNA Delivery

Binding of CC49-16K sFv/plasmid DNA complex to LS 174-T cells wasdemonstrated by in situ immunochemistry.

Experimental Protocol: LS174-T cells were grown in T75 tissue cultureflasks in MEM medium containing 1×non-essential amino acids, 1×Earles'salts and 1% fetal bovine serum at 37° C. with 5% CO₂. Cells (2×10⁶cells) in a T75 flask were treated with 1×trypsin/EDTA and split intofour T75 flasks and incubated 16 hrs. The experimental procedure is asfollows. (1) 8×10⁶ LS 174-T cells were collected by trypsin/EDTAdigestion from the 4 T75 flasks. (2) The cells were centrifuged at 3,000rpm at 4° C. (3) The supematant was discarded and the cells wereresuspended in PBS at 4° C. (4) The cells were centrifuged at 3,000 rpm,the supernatant discarded, and 2 ml of 1% paraformaldehyde (PFA) in PBSwas added to the pellet on ice for 30 min. (5) The cells were washedwith PBS, twice at 4° C. (6) The CC49-16K sFv and GX9251 samples wereanalyzed and quantitated by SDS-PAGE. (7) 5 μl (0.1 fg) of DIG-labeled(digoxigenin-labeled) pBR328 plasmid DNA (Boehringer Mannheim Cat.No.1585 738) was mixed with 200 μl of native GX9251 CC49/218 sFv (15μg/ml) or with 200 μl of EN266(7) CC49-16K sFv (approx. 5 μg/ml) at RTfor 30-min. (8) The LS174-T cells were added to the DIG-labeled pBR328plasmid/sFv mixture and incubated at RT for 30 min. (9) The cells werewashed twice with PBS. (10) The cells were centrifuged and resuspendedin 200 μl of PBS containing 1% BSA and a 1:100 dilution ofanti-digoxigenin-AP (alkaline phosphatase) Fab (Boehringer Mannheim Cat.No. 1093 274). Incubation was done at RT for 30 min. (11). The cellswere washed twice with PBS. (12). The cells were centrifuged and thepellet was resuspended in 100 μl of Fast Red solution (one tablet ofFast Red was dissolved in 500 μl of 0.1 M Tris-HCl, 0.15 M NaCl, pH 8.3;Fast Red Tablets are obtained from Boehringer Mannheim, Cat. No. 1 496549). Incubation was at RT for 30 min. (13). 50 μl of each sample werepipetted onto a glass slide, covered with a cover slip, and observedimmediately under a microscope (Nikon) using a 20×object lense andphotographed.

GX9251 CC49/218 sFv sample was used in the complex with the pBR328plasmid DNA. Background staining was minimal. EN266(7) CC49-16K sFvsample was used in the complex with the plasmid DNA. Positive redstaining was visually intense. Staining was more apparent in regions ofcell debris presumably due to the nature of the cell surface TAG-72antigen which is repetitive and easily shed making it more denselyconcentrated in these regions. Since the detection signal results fromthe presence of the DIG-labeled pBR328 plasmid DNA, this experimentdemonstrated (1) that the CC49-16K sFv can target plasmid to LS 174-Tcells but native CC49/218 sFv can not do so and (2) that the affinity ofthe plasmid for the CC49-16K sFv proteins is sufficient to remaincomplexed through several washing steps.

Example 6 Cell Transfection

This example demonstrates the transfection of LS 174-T cells by reporterplasmid pSEAP2 using CC49-16K sFv as carrier.

Protocol: The SEAP Reporter plasmid system (PT3057-2) was obtained fromClontech (Palo Alto, Calif.) and used according to the supplier'sinstructions. The pSEAP2 plasmid expresses a gene encoding a secretedalkaline phosphatase which serves as a reporter for successfultransfection of a cultured cell. The LIPOFECTAMINE PLUS reagent whichenhances transfection of DNA was obtained from Life Technologies(Gaithersburg, Md., Cat. No. 10964-013) and used according to thesupplier's instructions. As initial controls, DNA binding of theCC49-16K to pSEAP2 was demonstrated by Gel Shift experiments asdescribed in Examples 1 and 3 and a suitable sFv to plasmid ratio wasdetermined as stated below. Plasmid pSEAP2 was also shown to besuccessfully transfected into LS174-T cells by the Lipofectainine methodusing the recommended protocol. Furthermore, the AP reporterchemiluminescence signal could be quantitated by exposure to an X-rayfilm such that the strength of the signals (grains on the film) wereproportional to the amount of plasmid added over a 0-5 μg range.

The demonstration of CC49-16K sFv targeted transfection of LS174-T byplasmid pSEAP2 employed the following protocol. All test articles weredone in duplicate. (1) LS174-T cells (3×10⁶) were plated on each well ofa six well (Costar) plate in DMGM medium with 10% fetal bovine serum(FBS), at 37° C. with 5% CO₂ overnight. (2) The cells were washed withHBSS. (3) In separate microfuge tubes, (a) 5 μl (5 μg) of plasmid pSEAP2and 50 μl (about 50 μg) of EN266(7) CC49-16K sFv; OR (b) 5 μl (5 μg) ofplasmid pSEAP2 and 50 μl (about 50 μg) of EN234 CC49/218 native sFv; OR(c) 5 μl (5 μg) of plasmid pSEAP and 50 μl of water were mixed andincubated at room temperature for 30 min in 200 μl of DMEM medium. (4)The sFv/plasmid mixtures were added onto the LS174-T cells and incubatedat 37° C. for 60 min. (5) The cells were washed twice with HBSS. (6) 12μl of PLUS reagent (Life Technologies Lipofectamine Plus kit) were mixedwith 100 μl of DMEM medium and added onto the LS174-T cells, thenincubated at 37° C. for 30 min. (7) 8 μl of Lipofectamine was mixed into100 μl of DMEM and added to each well of the LS174-T plate, thenincubated at 37° C. for 30 min. (8) 0.8 ml of DMEM was added to eachwell and incubated at 37° C. for 3 hrs. (9) 100 μl of FBS and 1 ml ofDMGM with 10% FBS were added. Incubation continued at 37° C. with 5% CO₂for 2 days. (10) 1 ml of culture medium was transferred from each wellinto 1.5 ml microfuge tubes. (11) The cells were centrifuged in amicrofuge to pellet the cells and debris. (12) The supernatants fromeach tube were transferred into a Centricon 10 (Amicon Inc.) andconcentrated to a volume of 0.1 ml. (13)25 μl of each sample were mixedwith 75 μl of 1× dilution buffer (Clontech) and incubated at 65° C. for30 min. (14) The samples were cooled to room temperature and 100 μl ofassay buffer (Clontech) were added with incubation at room temperaturefor 5 min. (15) 100 μl of 1.25 mM CSPD with 1×chemiluminescence enhancer(Clontech) were mixed into each tube. (16) 150 μl of each sample weretransferred into individual wells of a DYNATECH microFLUOR plate. (17)The microtiter plate was overlayed with X-ray film and the film wasexposed for 3 hrs at room temperature.

The results are shown in FIGS. 7A and 7B. Lanes in the exposed x-rayfilm with duplicate lanes top and bottom are as follows. Lanes: 1.Positive control with 0.5 μl of pure placental alkaline phosphatase inoverexposed well; 2. Standard (Lipofectamine Plus) transfection ofpSEAP2 without sFv (i.e., condition c above) where washing steps (5)above are omitted; 3. LS174-T cell control with no added plasmid or sFv;4. pSEAP plasmid transfection without sFv (condition c above); 5. pSEAP2plasmid plus EN234 native sFv as described for condition b above; 6.pSEAP2 plasmid plus EN266(7) CC49-16K sFv as described for condition aabove; 7. Same as lane 6 except both protocol steps 5 (washings) and 7(Lipofectamine) are omitted; 8. Control with DMEM medium alone. FIGS. 7Aand 7B show the area quantitations of the x-ray film which werepreformed by densitometry scanning using a Molecular Dynamics PD-SIlaser scanner. Quantitation data are provided for both top (FIG. 7A;lane 8 was not scanned) and bottom (FIG. 7B; lanes 1 and 8 were notscanned) rows. Note that CC49-16K sFv (lane 6) promotes transfectionabout 8-fold over plasmid-alone control levels (lane 4) in thisexperiment.

Summary of transfection experiments: The area quantitation resultsdemonstrate that plasmid pSEAP can not efficiently transfect the cellsin the absence of CC49-16K sFv (lane 4). The plasmid is simply washedoff the cells in step 5. However, CC49-16K inclusion (lane 6) allows thesFv/plasmid complexes to remain attached to the cells and transfectionproceeds as efficiently as in a standard transfection protocol (lane 2)where the washing steps are omitted. Lane 5 shows that native sFv has aminor but detectable enhancement of transfection. This may be due tononspecific association of this very basic (pI˜9.3) sFv with thenegatively charged DNA. Lane 7 suggests that the CC49-16K/plasmidcomplex with no Lipofectamine added is slightly better than standard(+lipofectamine) transfection (with no washing at step 5).

Example 7 Synthesis of DNA Binding Regions in Other sFvs

Oligonucleotide-directed mutagenesis, synthetic linker ligation orpolymerase chain reaction can be employed to create oligo-lysine oroligo-arginine C-terminal tail in an sFv having a Kabat consensusV_(K)I/218/V_(H)III sFv (FIG. 8), C6.5/2 18 sFv (FIG. 9), and A33/218sFv. Amino acid assignments of the Kabat consensus V_(K)I/218/V_(H)IIIsFv and A33/218 sFv are according to Kabat et al., Sequences of proteinsof immunological Interest, pp. 108 & 331, 5th ed., U.S. Dept. Health andHuman Services, Bethesda, Md. (1991), where the assigned amino acidresidue at a position is the most commonly occurring amino acid at thatposition. Amino acid assignments of the wild-type C6.5 variable domainsare according to Schier, R., et al., J. Mol Biol. 255:28-43 (1996).

The mutated sFvs are individually ligated into the Pichia transferplasmid pHIL-S1 or pIC9 (Invitrogen Corp.) and transformed into Pichiapastoris. Detailed protocols for these procedures are presented in thePichia Expression Kit Instruction Manual Cat. No. X1710-01 (1994) fromInvitrogen Corporation. The sFv variants are placed behind a yeastsignal sequence in these constructions and the integrated sFv in theyeast transformants are tested for secretion of the sFv proteins.Evaluation of expression is done by Coomassie staining of SDS-PAGE gels.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those skilled in the art that various modifications can be madeto the disclosed embodiments and that such modifications are intended tobe within the scope of the present invention.

All documents, e.g., scientific publications, patents and patentpublications recited herein are hereby incorporated by reference intheir entirety to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by referencein its entirety. Where the document cited only provides the first pageof the document, the entire document is intended, including theremaining pages of the document.

13 1 782 DNA Artificial Sequence Description of Artificial SequenceCC49/218 sFv 1 gac gtc gtg atg tca cag tct cca tcc tcc cta cct gtg tcagtt ggc 48 Asp Val Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser ValGly 1 5 10 15 gag aag gtt act ttg agc tgc aag tcc agt cag agc ctt ttatat agt 96 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu TyrSer 20 25 30 ggt aat caa aag aac tac ttg gcc tgg tac cag cag aaa cca gggcag 144 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln35 40 45 tct cct aaa ctg ctg att tac tgg gca tcc gct agg gaa tct ggg gtc192 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 5055 60 cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc tcc240 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 6570 75 80 atc agc agt gtg aag act gaa gac ctg gca gtt tat tac tgt cag cag288 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 8590 95 tat tat agc tat ccc ctc acg ttc ggt gct ggg acc aag ctt gtg ctg336 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100105 110 aaa ggc tct act tcc ggt agc ggc aaa ccc ggg agt ggt gaa ggt agc384 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115120 125 act aaa ggt cag gtt cag ctg cag cag tct gac gct gag ttg gtg aaa432 Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 130135 140 cct ggg gct tca gtg aag att tcc tgc aag gct tct ggc tac acc ttc480 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145150 155 160 act gac cat gca att cac tgg gtg aaa cag aac cct gaa cag ggcctg 528 Thr Asp His Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu165 170 175 gaa tgg att gga tat ttt tct ccc gga aat gat gat ttt aaa tacaat 576 Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn180 185 190 gag agg ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tccagc 624 Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser195 200 205 act gcc tac gtg cag ctc aac agc ctg aca tct gag gat tct gcagtg 672 Thr Ala Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val210 215 220 tat ttc tgt aca aga tcc ctg aat atg gcc tac tgg ggt caa ggaacc 720 Tyr Phe Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr225 230 235 240 tcg gtc acc gtc tcc aaa aag aag aaa aaa aag aaa aag gtcacc gtc 768 Ser Val Thr Val Ser Lys Lys Lys Lys Lys Lys Lys Lys Val ThrVal 245 250 255 tcc taataggatc c 782 Ser 2 257 PRT Artificial SequenceDescription of Artificial Sequence CC49/218 sFv 2 Asp Val Val Met SerGln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15 Glu Lys Val ThrLeu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln LysAsn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys LeuLeu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg PheThr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 Ile Ser SerVal Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr SerTyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 Lys GlySer Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 ThrLys Gly Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150155 160 Thr Asp His Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu165 170 175 Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys TyrAsn 180 185 190 Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys SerSer Ser 195 200 205 Thr Ala Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu AspSer Ala Val 210 215 220 Tyr Phe Cys Thr Arg Ser Leu Asn Met Ala Tyr TrpGly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Lys Lys Lys Lys LysLys Lys Lys Val Thr Val 245 250 255 Ser 3 818 DNA Artificial SequenceDescription of Artificial Sequence CC49/218 sFv 3 gac gtc gtg atg tcacag tct cca tcc tcc cta cct gtg tca gtt ggc 48 Asp Val Val Met Ser GlnSer Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15 gag aag gtt act ttgagc tgc aag tcc agt cag agc ctt tta tat agt 96 Glu Lys Val Thr Leu SerCys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 ggt aat caa aag aac tacttg gcc tgg tac cag cag aaa cca ggg cag 144 Gly Asn Gln Lys Asn Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 tct cct aaa ctg ctg att tactgg gca tcc gct agg gaa tct ggg gtc 192 Ser Pro Lys Leu Leu Ile Tyr TrpAla Ser Ala Arg Glu Ser Gly Val 50 55 60 cct gat cgc ttc aca ggc agt ggatct ggg aca gat ttc act ctc tcc 240 Pro Asp Arg Phe Thr Gly Ser Gly SerGly Thr Asp Phe Thr Leu Ser 65 70 75 80 atc agc agt gtg aag act gaa gacctg gca gtt tat tac tgt cag cag 288 Ile Ser Ser Val Lys Thr Glu Asp LeuAla Val Tyr Tyr Cys Gln Gln 85 90 95 tat tat agc tat ccc ctc acg ttc ggtgct ggg acc aag ctt gtg ctg 336 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly AlaGly Thr Lys Leu Val Leu 100 105 110 aaa ggc tct act tcc ggt agc ggc aaaccc ggg agt ggt gaa ggt agc 384 Lys Gly Ser Thr Ser Gly Ser Gly Lys ProGly Ser Gly Glu Gly Ser 115 120 125 act aaa ggt cag gtt cag ctg cag cagtct gac gct gag ttg gtg aaa 432 Thr Lys Gly Gln Val Gln Leu Gln Gln SerAsp Ala Glu Leu Val Lys 130 135 140 cct ggg gct tca gtg aag att tcc tgcaag gct tct ggc tac acc ttc 480 Pro Gly Ala Ser Val Lys Ile Ser Cys LysAla Ser Gly Tyr Thr Phe 145 150 155 160 act gac cat gca att cac tgg gtgaaa cag aac cct gaa cag ggc ctg 528 Thr Asp His Ala Ile His Trp Val LysGln Asn Pro Glu Gln Gly Leu 165 170 175 gaa tgg att gga tat ttt tct cccgga aat gat gat ttt aaa tac aat 576 Glu Trp Ile Gly Tyr Phe Ser Pro GlyAsn Asp Asp Phe Lys Tyr Asn 180 185 190 gag agg ttc aag ggc aag gcc acactg act gca gac aaa tcc tcc agc 624 Glu Arg Phe Lys Gly Lys Ala Thr LeuThr Ala Asp Lys Ser Ser Ser 195 200 205 act gcc tac gtg cag ctc aac agcctg aca tct gag gat tct gca gtg 672 Thr Ala Tyr Val Gln Leu Asn Ser LeuThr Ser Glu Asp Ser Ala Val 210 215 220 tat ttc tgt aca aga tcc ctg aatatg gcc tac tgg ggt caa gga acc 720 Tyr Phe Cys Thr Arg Ser Leu Asn MetAla Tyr Trp Gly Gln Gly Thr 225 230 235 240 tcg gtc acc gtc tcc aaa aagaag aaa aaa aag aaa aag gtc acc gtc 768 Ser Val Thr Val Ser Lys Lys LysLys Lys Lys Lys Lys Val Thr Val 245 250 255 tcc aaa aag aag aaa aaa aagaaa aag gtc acc gtc tcc taataggatc c 818 Ser Lys Lys Lys Lys Lys Lys LysLys Val Thr Val Ser 260 265 4 269 PRT Artificial Sequence Description ofArtificial Sequence CC49/218 sFv 4 Asp Val Val Met Ser Gln Ser Pro SerSer Leu Pro Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Leu Ser Cys LysSer Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu AlaTrp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr TrpAla Ser Ala Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser GlySer Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 Ile Ser Ser Val Lys Thr GluAsp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu ThrPhe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 Lys Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Gln ValGln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140 Pro Gly Ala SerVal Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 Thr AspHis Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu 165 170 175 GluTrp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 195 200205 Thr Ala Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 210215 220 Tyr Phe Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr225 230 235 240 Ser Val Thr Val Ser Lys Lys Lys Lys Lys Lys Lys Lys ValThr Val 245 250 255 Ser Lys Lys Lys Lys Lys Lys Lys Lys Val Thr Val Ser260 265 5 265 PRT Artificial Sequence Description of Artificial SequenceA33/218 sFv 5 Asp Val Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr SerVal Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn ValArg Thr Val 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro LysThr Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp ArgPhe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser AsnVal Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His TrpSer Tyr Pro Leu 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Val Lys GlySer Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser ThrLys Gly Glu Val Lys 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val LysPro Gly Gly Ser Leu Lys 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe AlaPhe Ser Thr Tyr Asp Met Ser 145 150 155 160 Trp Val Arg Gln Thr Pro GluLys Arg Leu Glu Trp Val Ala Thr Ile 165 170 175 Ser Ser Gly Gly Ser TyrThr Tyr Tyr Leu Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser ArgAsp Ser Ala Arg Asn Thr Leu Tyr Leu Gln Met 195 200 205 Ser Ser Leu ArgSer Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Pro Thr 210 215 220 Thr Val ValPro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 SerLys Lys Lys Lys Lys Lys Lys Lys Val Thr Val Ser Lys Lys Lys 245 250 255Lys Lys Lys Lys Lys Val Thr Val Ser 260 265 6 283 PRT ArtificialSequence Description of Artificial Sequence Kabat Consensus 6 Asp IleGln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 AspArg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Leu Val Ser Ile 20 25 30 SerAsn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 LeuLeu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg 50 55 60 PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser 85 90 95Leu Pro Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly 100 105110 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 115120 125 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly130 135 140 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerSer 145 150 155 160 Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys GlyLeu Glu Trp 165 170 175 Val Ser Val Ile Ser Gly Lys Thr Asp Gly Gly SerThr Tyr Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn 195 200 205 Thr Leu Tyr Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Gly Arg Xaa GlyXaa Ser Leu Ser Gly Xaa Tyr 225 230 235 240 Tyr Tyr Tyr His Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255 Val Ser Ser Lys Lys LysLys Lys Lys Lys Lys Val Thr Val Ser Lys 260 265 270 Lys Lys Lys Lys LysLys Lys Val Thr Val Ser 275 280 7 282 PRT Artificial SequenceDescription of Artificial Sequence C6.5/218 sFv 7 Gln Ser Val Leu ThrGln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr IleSer Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser TrpTyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly HisThr Asn Arg Pro Ala Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys SerGly Thr Ser Ala Ser Leu Ala Ile Ser Gly Phe Arg 65 70 75 80 Ser Glu AspGlu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly TrpVal Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 100 105 110 Ser ThrSer Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 115 120 125 GlyGln Val Gln Leu Leu Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly 130 135 140Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser 145 150155 160 Tyr Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Tyr165 170 175 Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser ProSer 180 185 190 Phe Gln Gly Gln Val Thr Ile Ser Val Asp Lys Ser Val SerThr Ala 195 200 205 Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser Asp Ser AlaVal Tyr Phe 210 215 220 Cys Ala Arg His Asp Val Gly Tyr Cys Ser Ser SerAsn Cys Ala Lys 225 230 235 240 Trp Pro Glu Tyr Phe Gln His Trp Gly GlnGly Thr Leu Val Thr Val 245 250 255 Ser Ser Lys Lys Lys Lys Lys Lys LysLys Val Thr Val Ser Lys Lys 260 265 270 Lys Lys Lys Lys Lys Lys Val ThrVal Ser 275 280 8 84 PRT Artificial Sequence Description of ArtificialSequence Nucleic acid binding region 8 Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 Lys Lys Lys Lys Lys Lys LysLys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Lys Lys Lys Lys 65 70 75 80 Lys Lys Lys Lys 9 84 PRTArtificial Sequence Description of Artificial Sequence Nucleic acidbinding region 9 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg ArgArg Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg ArgArg Arg Arg 20 25 30 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg ArgArg Arg Arg 35 40 45 Arg Arg Arg Arg Arg Arg Arg Arg Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa ArgArg Arg Arg 65 70 75 80 Arg Arg Arg Arg 10 83 PRT Artificial SequenceDescription of Artificial Sequence Nucleic acid binding region 10 ArgLys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys 1 5 10 15Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys 20 25 30Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys 35 40 45Arg Lys Arg Lys Arg Lys Arg Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys Arg Lys 65 70 7580 Arg Lys Arg 11 84 PRT Artificial Sequence Description of ArtificialSequence Nucleic acid binding region 11 Arg Arg Arg Arg Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30 Arg Arg Arg Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45 Arg Arg Arg Arg Arg Arg ArgArg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Lys Lys Lys Lys 65 70 75 80 Lys Lys Lys Lys 12 36DNA Artificial Sequence Description of Artificial SequenceOligonucleotide 12 gtcaccgtct ccaaaaagaa gaaaaaaaag aaaaag 36 13 36 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide13 gtgacctttt tctttttttt cttctttttg aagacg 36

What is claimed is:
 1. A genetically engineered single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking the first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein at its C-terminus, N-terminus, or both,the single-chain antigen-binding polypeptide comprises basic amino acidresidues selected from the group consisting of: oligo-Lys, oligo-Arg anda combination thereof.
 2. The single-chain antigen-binding polypeptideof claim 1, wherein said first polypeptide (a) comprises the antigenbinding portion of the variable region of an antibody light chain andsaid second polypeptide (b) comprises the antigen binding portion of thevariable region of an antibody heavy chain.
 3. The single-chainantigen-binding polypeptide of claim 1, wherein the C-terminus of saidsecond polypeptide (b) comprises a deletion of one or plurality of aminoacid residue(s), such that the remaining N-terminus amino acid residuesof the second polypeptide are sufficient for the single-chainantigen-binding polypeptide to be capable of binding an antigen.
 4. Thesingle-chain antigen-binding polypeptide of claim 1, wherein the basicamino acid residues of the single-chain antigen-binding polypeptidecomprises at least 2 groups of eight consecutive Lys residues, whereineach group of eight consecutive Lys residues is separated from theadjacent group by 0-20 amino acid residues.
 5. The single-chainantigen-binding polypeptide of claim 1, wherein the basic amino acidresidues comprises at least 2 groups of eight consecutive Arg residues,wherein each group of eight consecutive Arg residues is separated fromthe adjacent group by 0-20 amino acid residues.
 6. The single-chainantigen-binding polypeptide of claim 1, wherein the basic amino acidresidues comprises at least 2 groups of eight consecutive residuesconsisting of Lys and Arg residues, wherein each group of eightconsecutive Lys and Arg residues is separated from the adjacent group by0-20 amino acid residues.
 7. The single-chain antigen-bindingpolypeptide of claim 1, wherein the basic amino acid residues comprisesat least 2 groups of eight consecutive Lys residues, Arg residues or acombination thereof, wherein each group of eight consecutive Lysresidues, Arg residues or combination thereof is separated from theadjacent group by 0-20 amino acid residues.
 8. The single-chainantigen-binding polypeptide of claim 7, wherein the basic amino acidresidues comprises 2 to 8 groups of eight consecutive Lys residues, Argresidues or a combination thereof, wherein each group of eightconsecutive Lys residues, Arg residues or combination thereof isseparated from the adjacent group by 0-20 amino acid residues.
 9. Thesingle-chain antigen-binding polypeptide of claim 8, wherein the basicamino acid residues comprises 2 to 6 groups of eight consecutive Lysresidues, Arg residues or a combination thereof, wherein each group ofeight consecutive Lys residues, Arg residues or combination thereof isseparated from the adjacent group by 0-20 amino acid residues.
 10. Thesingle-chain antigen-binding polypeptide of claim 9, wherein the basicamino acid residues comprises 2 to 4 groups of eight consecutive Lysresidues, Arg residues or a combination thereof, wherein each group ofeight consecutive Lys residues, Arg residues or combination thereof isseparated from the adjacent group by 0-20 amino acid residues.
 11. Thesingle-chain antigen-binding polypeptide of claim 10, wherein the basicamino acid residues comprises 2 to 3 groups of eight consecutive Lysresidues, Arg residues or a combination thereof, wherein each group ofeight consecutive Lys residues, Arg residues or combination thereof isseparated from the adjacent group by 0-20 amino acid residues.
 12. Amultivalent single-chain antigen-binding protein, comprising two or moregenetically engineered single-chain antigen-binding polypeptides, eachgenetically engineered single-chain antigen-binding polypeptidecomprising: (a) a first polypeptide comprising the antigen bindingportion of the variable region of an antibody heavy or light chain; (b)a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and (c) a peptidelinker linking the first and second polypeptides (a) and (b) into asingle chain polypeptide having an antigen binding site, wherein at itsC-terminus, N-terminus, or both, the single-chain antigen-bindingpolypeptide comprises basic amino acid residues selected from the groupconsisting of: oligo-Lys, olio-Arg and a combination thereof.
 13. Themultivalent protein of claim 12, wherein said first polypeptide (a)comprises the antigen binding portion of the variable region of anantibody light chain and said second polypeptide (b) comprises theantigen binding portion of the variable region of an antibody heavychain.
 14. The multivalent protein of claim 12, wherein the C-terminusof said second polypeptide (b) comprises a deletion of one or pluralityof amino acid residue(s), such that the remaining N-terminus amino acidresidues of the second polypeptide are sufficient for the single-chainantigen-binding polypeptide to be capable of binding an antigen.
 15. Themultivalent single-chain antigen-binding protein of claim 12, whereinthe basic amino acid residues of the single-chain antigen-bindingpolypeptide comprises at least 2 groups of eight consecutive Lysresidues, wherein each group of eight consecutive Lys residues isseparated from the adjacent group by 0-20 amino acid residues.
 16. Themultivalent single-chain antigen-binding protein of claim 12, whereinthe basic amino acid residues comprises at least 2 groups of eightconsecutive Arg residues, wherein each group of eight consecutive Argresidues is separated from the adjacent group by 0-20 amino acidresidues.
 17. The multivalent single-chain antigen-binding protein ofclaim 12, wherein the basic amino acid residues comprises at least 2groups of eight consecutive residues consisting of Lys and Arg residues,wherein each group of eight consecutive Lys and ARG residues isseparated from the adjacent group by 0-20 amino acid residues.
 18. Themultivalent single-chain antigen-binding polypeptide of claim 12,wherein the basic amino acid residues comprises at least 2 groups ofeight consecutive Lys residues, Arg residues or a combination thereof,wherein each group of eight consecutive Lys residues, Arg residues orcombination thereof is separated from the adjacent group by 0-20 aminoacid residues.
 19. The multivalent single-chain antigen-bindingpolypeptide of claim 18, wherein the basic amino acid residues comprises2 to 8 groups of eight consecutive Lys residues, Arg residues or acombination thereof, wherein each group of eight consecutive Lysresidues, Arg residues or combination thereof is separated from theadjacent group by 0-20 amino acid residues.
 20. The multivalentsingle-chain antigen-binding polypeptide of claim 19, wherein the basicamino acid residues comprises 2 to 6 groups of eight consecutive Lysresidues, Arg residues or a combination thereof, wherein each group ofeight consecutive Lys residues, Arg residues or combination thereof isseparated from the adjacent group by 0-20 amino acid residues.
 21. Themultivalent single-chain antigen-binding polypeptide of claim 20,wherein the basic amino acid residues comprises 2 to 4 groups of eightconsecutive Lys residues, Arg residues or a combination thereof, whereineach group of eight consecutive Lys residues, Arg residues orcombination thereof is separated from the adjacent group by 0-20 aminoacid residues.
 22. The multivalent single-chain antigen-bindingpolypeptide of claim 21, wherein the basic amino acid residues comprises2 to 3 groups of eight consecutive Lys residues, Arg residues or acombination thereof, wherein each group of eight consecutive Lysresidues, Arg residues or combination thereof is separated from theadjacent group by 0-20 amino acid residues.